Metabolic profile of veins and their implications in primary varicose veins Disease.

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1 Metabolic profile of veins and their implications in primary varicose veins Disease. Anwar MA 1, Beckonert OP 2, Shalhoub J 1, Vorkas P 2, Lim CS 1, Want EJ 2, Nicholson JK 2, Holmes E 2, Davies AH 1 1 Academic Section of Vascular Surgery 2 Section of Biomolecular Medicine Department of Surgery & Cancer Imperial College London

2 Background Stresses to the vein wall (hypoxia, mechanical stretch & low shear stress) PredisposingRisk factors Genetic, age, sex, pregnancy,?obesity,?poor mobility Blood stasis (Venous hypertension) Vein wall changes(endothelial injury), Venous reflux Valve Valvular incompetence Vein wall Lim and Davies, Br J Surg, 2009 Lumen Vein wall weakening and relaxation

3 Background Intimal hyperplasia, areas of hypertropy and atrophy Smooth muscle cell proliferation, dysregulated apoptosis and migration Matrix remodelling and collagen disruption with MMP/TIMP imbalances Cario-Toumaniantz et al. J Vasc Res, 2007 Lim and Davies, Br J Surg, 2009 Lee et al. Dermatol Surg, 2005

4 Background Transcriptomics Gene / RNA Protein Proteomics Metabonomics Metabolites Metabonomics is the quantitative measurement of the dynamic multi-parametric metabolic response of living systems to pathophysiological stimuli. Nicholson and Holmes, Xenobiotica, 1999

5 Nuclear Magnetic Resonance Identification of biomarkers Spectroscopy Elucidation of cellular pathways Identification of drug efficacy and toxicity Gene and enzyme functions Targeting therapeutics Lindon, Holmes and Nicholson, Pharm Res, Yap et al. INTERMAP study, J Proteome Res, 2010 Nicholson et al. Nat Rev Drug Discov, 2002 Chan et al. J Proteome Res, 2009 Brindle et al. Nat Med, 2002

6 Aims & Objectives This pilot study aims to identify the distinct metabolic profiles of intact varicose veins with a view to promoting the understanding of the pathogenesis of varicose vein formation.

7 Selection of Control Veins Standard control veins for studies using human varicose veins for analysis -Great Saphenous Vein (GSV) used for bypass operation Other options Facial veins Inferior Epigastric Vein Davies et al, EJVES, 2012 Lim et al, JVS, 2012 Raffetto et al, JVS, 2009

8 Demographics Varicose GSV Non-Varicose GSV Facial Veins Number of patients Age, mean (range) years 43 (31-62) 69 (32-94) 67 (47-75) Gender 6 Male / 2 Female 7 Male / 1 Female 2 Male/ 3 Female Smoker Ischemic heart disease Hypertension Peripheral arterial disease Diabetes 0 3 0

9 Varicose Veins Methods Control Veins Varicose GSV n=8 Facial Veins n=5 Non-Varicose GSV n=8 Entire circumference of vein tissue (average weight /- 0.8 grams) 1D/2D 1 H NMR 600 MHZ, MAS=54.7 Statistical Analysis Beckonert et al. Nat Protoc, 2010

10 Variation in the Vein Samples Without Class Discrimination Non-Varicose GSV Facial Veins Varicose Veins t[2] t[2] t[1] t[1] Principal Component Analysis

11 Comparison of Varicose and Non Varicose Veins Based on their Discriminatory Metabolic Profiles Non-Varicose GSV Facial Veins Varicose Veins t[2] Orthogonal Partial Least Square Analysis t[1]

12 Non-Varicose Veins Spectra Varicose Veins Spectra Alanine β-glucose Glucose Glycogen Isoleucine Leucine Triglycerides Tyrosine Phenylalanine Valine Alanine β-glucose Creatine Glucose Glycogen Glutamate Glutathione Isoleucine Lactate Myo-inositol Leucine Threonine Triglyceride

13 Non-Varicose GSV Spectra Facial Veins Spectra

14 Influential Metabolites Responsible for the Separation between Varicose and Non- Varicose Veins n=8 n=13 Orthogonal-2 Partial Least Squares Analysis

15 Implications Creatine Phosphate Creatine Hypoxia Glucose ATP ADP HIF-1α Glycolysis Pyruvate Lactic Acid Phosphatidylinositides (2 nd Messengers) Intracellular Signalling PKC PI3K NFκB Inflammation Growth/ Apoptosis Matrix Metalloproteinases in vascular cells

16 Conclusion The metabolic profile of varicose veins is different from non-varicose veins

17 Acknowledgements Graham-Dixon Charitable Trust

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