Panagiotis Vorkas (Presenter)
Imperial College London
Bio: Panos is a post-doctoral researcher at the Section of Computational and Systems Medicine of Imperial College London. He obtained a Bachelor degree in Chemistry in 2006 and a Masters in Clinical Chemistry in 2009. He was awarded a PhD in Biochemistry in August 2014. He is focusing in bioanalysis with special interest in developing untargeted metabolic and lipid profiling approaches, with the ultimate aim of expanding metabolome coverage. He is working under the theme of cardiovascular disease and risk factors. Guided by biomarker discovery approaches he is now elaborating on biomarker validation and elucidation of disease pathways.
Authorship: Panagiotis A Vorkas (1), Shamim Rahman (2) , Alun H Davies (3), Elaine Holmes (1), Kevin J Woollard (2)
(1) Biomolecular Medicine, Division of Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, UK (2) Division of Immunology and Inflammation, Department of Medicine, Faculty of Medicine, Imperial College London, UK (3) Academic Section of Vascular Surgery, Division of Surgery, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, UK
| Short Abstract Atherosclerosis remains the leading cause of mortality and morbidity in the western world. Here, UPLC-MS-based metabonomics were utilized for the analysis of human atherosclerotic tissue. A panel of established pathways were identified being dysregulated, such as free cholesterol (FC), 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 disease. PE-Cer also demonstrated the highest inverse correlation to FC. Pilot studies in primary human monocyte-derived macrophage foam cells demonstrated elevated apoptosis, accompanied by 2-fold reduction of SAMD8, the enzyme responsible for PE-Cer synthesis. This provides insight for the role of PE-Cer in foam cell formation and atherogenesis. |
Long Abstract
Introduction
Atherosclerosis is the number one cause of mortality and morbidity in the western world. Most adverse health events associated with atherosclerosis are due to flow limitation because of advanced stenosing plaque or plaque rupture which can lead to distant embolization and ischemia. Additionally, existing pharmaceutical schemes have had limited success in primary prevention, while the scientific community still struggles to find appropriate dose, drug regimes, and mechanisms of action.
In this study, we adopted two UPLC-MS-based metabolic profiling approaches with subsequent computational modelling and statistics, to study the metabolic changes of the progression from intimal thickening (INT) - the immediate pre-plaque stage, as a control - to the formation of the atherosclerotic plaque. Plaques were obtained from the carotid (CAR) and femoral (FEM) arterial bifurcations. Adjacent INT tissue, was obtained from the proximal and distal extends of the atherosclerotic lesion. The use of INT as control (rather the normal tissue) comes with the advantage of being the immediate (benign) pre-plaque stage, providing a more realistic course of the disease and focusing on the biological pathways solely responsible for lipid deposition. The employed UPLC-MS methodologies utilized reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). Using highly predictive and cross-validated multivariate statistical models and univariate statistics, we show metabolites dysregulated in plaque tissue as compared to INT. The strongest dysregulations were used in order to generate a novel hypothesis, which was further verified in vitro.
Methods
The 96 tissue samples (CAR: n=52, FEM: n=26, INT: n=16) from 78 patients were subjected to tissue homogenization and metabolite extraction using two consecutive extractions: polar (Aqueous Extract, AQ), and organic (Organic Extract, ORG). UPLC-MS analyses were conducted using the Acquity UPLC system with BEH HILIC column for AQ and CSH for ORG (Waters Ltd). Detection was conducted in both polarities using Q-TOF Premier and Xevo G2 Q-TOF Mass Spectrometers (Waters MS Technologies), respectively. Metabolic features were extracted using MarkerLynx package. Multivariate statistics were performed using SIMCA-P+12 software. Two-tailed t-test was performed assuming unequal variance. Metabolite identification was assisted by database m/z matching, isotopic pattern, MS/MS and MSE spectra, and matching to authentic standards. Pathway analysis included mapping of metabolites to biological pathways using KEGG database, and Spearman pairwise correlation analysis.
In vitro studies were performed using primary human foam cells derived from monocyte-derived macrophages. Peripheral blood monocytes were isolated healthy volunteers. After one-week treatment with macrophage colony stimulating factor (M-CSF), monocyte-derived macrophages were treated with vehicle and different combinations of oxLDL, soluble free cholesterol and Sandoz 58-035 (an acyl-CoA:cholesterol acyltransferase inhibitor). Using the vehicle and oxLDL treatments as controls. RNA was isolated from treated cells and SAMD8 mRNA was quantified using quantitative RT-PCR and analysed using the ΔΔCt method against house keeping genes. Additionally, a flow cytometry cell death assay was employed to measure the levels of apoptosis and necrosis.
Results and Discussion
By using a combination of multivariate and univariate statistics the metabotype of the lipid-laden plaque was revealed. This task encompassed structural assignment of >150 metabolites. All three analyzed groups (INT, CAR and FEM) demonstrated distinct profiles using multivariate statistics. However, several metabolites were commonly dysregulated in plaque tissue from both anatomical locations. This common signature of perturbed metabolites can provide a good overview of the basis of metabolic dysregulation leading to plaque formation.
A combination of established for their involvement in atherosclerosis metabolites were detected being dysregulated in plaques attributing validity to the chosen experimental approach. Highlights of these findings were the higher intensities of free cholesterol and several oxidized cholesterol ester species, as well as cholesterol sulphate. Purine and pyrimide intensities were also detected reduced in plaques. These included uridine, inosine, hypoxanthine, guanosine and methyluridine, which are known for their involvement in inflammation and vascular tone. Additionally, a known pathway involved in apoptosis, the ceramide pathway, appeared to suffer complete loss of homeostasis in atherosclerosis. This was detected by a reduction in several ceramides (Cer) such as Cer(d18:1/22:0) and Cer(d18:1/23:0), sphingosine, C16-sphingosine, and two hexosylceramides (HexCer), specifically HexCer(d18:1/23:0) and HexCer(d18:1/24:0). On the other hand, several sphingomyelin moieties, as well as tri- and tetraHexCer(d18:1/16:0) were detected in lower intensities in the diseased groups. Lastly, truncation of β-oxidation was identified by the accumulation of long- and medium-chain acylcarnitines and reduction of short-chain acylcarnitines in the diseased groups. This finding along with the consumption of unsaturated fatty acids reveals a dysregulation of metabolic oxidation.
A novel finding of this study was the detected lower levels of phosphatidylethanolamine-ceramides (PE-Cer) by 2-fold in the atherosclerotic plaques. This lipid class has not been previously investigated in the context of atherosclerosis. PE-Cers are known to be members of the Cer pathway, however detected in low concentrations in humans. Two PE-Cer species were detected and with high statistical significance, namely PE-Cer(d18:1/16:0) and PE-Cer(d18:1/24:1). PE-Cer(d18:1/16:0) was detected with t-test p-value as low as 9.8 x 10-12 when FEM were compared to INT. Most importantly, these metabolites manifested high and inverse Spearman pairwise correlations to free cholesterol (r=-0.76 and r=-0.61). This inverse association to free cholesterol was higher than any other detected metabolite, even from the remainder of the Cer pathway as well as oxidized cholesteryl esters. These findings led as to assume a mechanistic approach for interpretation.
It is nowadays established that free cholesterol loading of macrophage cells can lead to apoptosis.1 Additionally, the enzyme responsible for PE-Cer synthesis, namely sphingomyelin synthase-related (SMSr), also known as sterile alpha domain 8 (SAMD8), has also been recently shown to be involved in ceramide induced mitochondrial apoptosis.2 It was therefore hypothesised that free cholesterol loading can lead to macrophage foam cell apoptosis via the PE-Cer synthesis pathway. Studies using free cholesterol loaded foam cells demonstrated elevated apoptosis, accompanied by a 2-fold reduction of the mRNA of the SMSr enzyme.
Conclusions
Overall, these findings demonstrate a novel pathway with potential involvement in atherogenesis, which was further validated using in vitro cell culture of macrophage foam cells. Our findings bear the potential for providing a novel pharmaceutical target. Additionally, PE-Cer detection in blood could be employed for disease diagnosis and tailoring atherosclerosis treatment.
References & Acknowledgements:
References
1. Yao PM and Tabas I. Free cholesterol loading of macrophages induces apoptosis involving the fas pathway. J Biol Chem. 2000;275:23807-13.
2. Tafesse FG, Vacaru AM, Bosma EF, Hermansson M, Jain A, Hilderink A, Somerharju P and Holthuis JC. Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis. J Cell Sci. 2014;127:445-54.
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