Characterisation of Biological Factors in the Pathogenesis of Varicose Veins

Abstract

Varicose veins affect one third of adults. Complications of varicose veins can have detrimental effects on the patient’s quality of life. Morphological changes including intimal hyperplasia, smooth muscle cells hypertrophy or atrophy and irregularities of extracellular matrix contents have demarcated varicose from non-varicose veins. Some of the biological mechanisms behind these changes have been illustrated by the studies investigating the individual expressions of proteins (proteomic) and genes (transcriptomics). The evidence from these studies also suggests the role of abnormal cell metabolism in the development of varicose veins. However, both transcriptomic and proteomic approaches are not able to fully explain the irregularities of cell metabolism. Metabolic profiling of tissue and biological fluids using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has the ability to elucidate irregular cell metabolic pathways and functions of genes and enzymes in disease pathogenesis. Metabolic profiling approaches are also used to identify toxicity and therapeutic efficacy of drugs.

Aims To identify the metabolic profile of varicose veins as compared to non-varicose veins with an aim to promote our understanding of the disease pathogenesis.

Methods Ethical approval was obtained from the local ethics committee. Firstly, a preliminary study was performed. Varicose veins (n=8) were removed from patients following varicose vein surgery. Non-varicose veins were removed from patients following operations where there was a removal of vein involved as a part of the procedure. Non-varicose veins tissue retrieved included great saphenous vein (n=8) from bypass or amputation. Facial veins (n=5) during the carotid endarterectomy were also removed. Metabolic profile of the intact varicose and non-varicose veins tissues were measured using 1D magic angle spinning (MAS) 1H NMR (600). In order to develop the most optimal organic and aqueous extraction method for vein tissues, 11 grams of varicose vein tissues retrieved from different patients was mixed using mortar and pestle and cryogenic impact mill in liquid nitrogen producing a homogenous tissue mixture. In total, 70 samples were prepared from this homogenate each having a weight of 145+/- 5 mg. For extraction of aqueous metabolites, 20 samples were treated with two different solvents concentrations: 10 samples with Water: Methanol (1:1), and 10 samples with Water: Methanol (3:1). For optimisation of organic metabolites, remaining 50 samples were extracted using 5 different organic solvents including dichloromethane, chloroform, isopropanol, hexane and ether. Each group had 10 samples or replicates. All aqueous and organic extracts were run on 1H NMR (600 MHz). Organic extracts were analyzed on ultra performance liquid chromatography mass spectrometry (UPLC-MS). Ex-vivo rat inferior vena cava stretch experiment was performed in vascular research laboratory, Harvard Medical School, Brigham and Women’s Hospital, US. Inferior vena cava of the male Sprague dowley rat (n=5) was divided into 4 segments. Two out of these 4 segments were kept at a basal tension of 0.5 gram for 4 and 18 hours and the remaining two were stretched with 2 grams weight for 4 and 18 hours in an organ culture bath. Aqueous extracts from rat IVC segments were analysed using H1 NMR (800 MHz). Organic extracts were analyzed on ultra performance liquid chromatography mass spectrometry (UPLC-MS).

Lastly, comprehensive profiling of human varicose veins (n=80) and non-varicose veins (n=35) tissue extracts was performed. The most optimal extraction method employed to extracts metabolites from human veins tissue was developed as mentioned earlier and the same extraction protocol was used to extracts metabolites from human veins tissues. Aqueous extracts were run on 1H NMR (600 MHz). Organic extracts were analyzed on ultra performance liquid chromatography mass spectrometry (UPLC-MS).

Spectra obtained from NMR for all experiments were mathematically modeled and statistically analyzed using chemomeric software including MATLAB and SIMCA. Metabolites were assigned using human metabolome databases and previous published reports. Metabolite identification was confirmed using 2D NMR. UPLC-MS based profiling data was analysed using MassLynx version 4.1 and SIMCA. UPLC-MS based metabolites were assigned using MS/MS experiment and with support databases including lipodomics and human metabolome database (HMDB). Pathway analysis was performed using ingenuity database and published reports. Multiple testing corrections using Benjamini Yakatieli method was performed to exclude false discovery rate in the data. Pathways analysis was performed using ingenuity and KEGG pathways databases.

Results Magic angle spinning NMR analysis of intact vein tissues showed the presence of lipid metabolites at a higher concentration in the non-varicose vein group whilst creatine, lactate, myo-inositol, and glutamate metabolites were more characteristic of the varicose vein group. The orthogonal partial least square (OPLS) coefficient plot revealed that the differential abundance of creatine, myo-inositol and lactate was highly correlated to varicose vein group. Metabolic profiles of facial veins were also different from varicose veins. Abundance of lipids was also noticed in facial veins. The most optimal method for human veins tissue extraction was found to be the consecutive approach of metabolites extraction via first extraction of organic metabolites with MTBE: methanol (3:1) followed by methanol: water (1:1) in terms of reproducibility and sensitivity. Metabolic differences in rat IVC segments were observed between rat IVC segments stretch for 18 hrs as compared to non-stretched for 18 hrs. Metabolites including choline, valine and triglycerides were found in high concentrations in stretched for 18 hrs group as compared to non-stretched for 18 hrs.

Comprehensive metabolic profiling of veins tissue extracts using two metabolomics analytical platforms (NMR and UPLC-MS) was determined. Both organic and aqueous metabolites were extracted from vein tissues using the most optimal extraction method developed above. Multivariate analysis of the NMR data from 80 varicose veins and 35 non-varicose veins showed glutamate, taurine and myo-inositol metabolites in higher concentration in varicose veins wall as compared to non-varicose veins wall. Multivariate analysis of the lipid metabolites revealed significantly increased concentrations of phosphatidylcholine (PC) and sphingomyelin (SM) in varicose veins as compared to non-varicose veins. Pathway analysis based on online databases and published reports showed association of myo-inositol with intracellular pathways linked to cellular proliferation and PC and SM with induction of inflammatory response.

Conclusions and Future Work This novel work demonstrates a comprehensive metabolic profile of human varicose veins with metabolites including PC, SM, myo-inositol, glutamate and taurine differentially associated with varicose veins. This work unravels new cellular pathways which should be the focus of future research and may enable us to understand disease pathogenesis in more details and identify therapeutic targets. Moreover, this work first time provides a comprehensive extraction methodology for human vein tissue metabolites extractions comprising of MTBE: methanol (3:1) followed by water: methanol (1:1). This work also showed that prolonged stretch of 18 hrs duration changes metabolic profile of rat IVC segments.