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Abstract Introduction
Atherosclerosis remains a leading worldwide cause of mortality and morbidity. Advanced stenosing plaque can result to flow limitation or plaque rupture, leading to ischemic stroke or heart attack.
Objectives
The primary objective of this study is to provide a comprehensive in-depth elucidation of the metabolic dysregulations associated with atherosclerotic plaque deposition and identify potential novel targets and biomarkers for diagnosis and treatment tailoring.
Methods
Ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS)-based metabolomics were utilized for the analysis of human advanced atherosclerotic tissue. From 78 patients, a total of 52 carotid and 26 femoral plaques were compared to 16 adjacent arterial non-plaque tissue (intimal thickening). Tissue samples were homogenised and extracted consecutively for aqueous and organic extracts. Aqueous extracts were analysed using hydrophilic interaction chromatography (HILIC-)UHPLC-MS, whilst organic extracts by reversed-phase (RP-)UHPLC-MS.
In vitro studies were performed using peripheral blood monocyte-derived macrophages (MDM) (from healthy volunteers). After one-week treatment with macrophage colony stimulating factor, MDM were treated with vehicle, acetylated-LDL (acLDL) and a combination of acLDL, soluble free unesterified cholesterol (FUEC) and Sandoz 58-035 (an acyl-CoA:cholesterol acyltransferase inhibitor). Using the vehicle and acLDL treatments as controls, the SAMD8 (the enzyme responsible for PE-Cer synthesis) gene mRNA was relatively quantified using real-time RT-PCR. Additionally, a flow cytometry cell death assay was employed to measure the levels of apoptosis and necrosis. Finally, cells from different treatments were extracted using organic solvents and analysed using a lipid profiling method (as described in the preceding paragraph).
Results
A panel of established as well as novel molecules, from several biological pathways, were identified as being dysregulated. These included FUEC, oxidized cholesteryl esters, purines, pyrimidines, sphingolipids and acylcarnitines. A previously unassociated sphingolipid, namely phosphatidylethanolamine-ceramide (PE-Cer), was detected with high statistical significance (p=9.8x10-12) and 2-fold reduction in plaque tissue. PE-Cer also demonstrated the highest (inverse) correlation to FUEC (ρ=-0.76).
In pilot validation studies, the acLDL/FUEC-treated MDM demonstrated elevated apoptosis, and a 2-fold reduction in PE-Cer, in concordance with the findings in human tissue. This was accompanied by a reduction of SAMD8 RNA. Finally, a comprehensive examination of the sphingolipid pathway demonstrated an increase in de novo ceramide synthesis, further to the recognised in apoptosis hydrolysis of sphingomyelin (to ceramide).
Conclusion
The PE-Cer pathway demonstrates a potentially pivotal role in advanced atherosclerosis, while previously unrecognised sphingolipid pathway alterations are revealed. |
= Discovery stage.
= Translation stage.
= Clinically available.
