A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis.

Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz 1H Nuclear Magnetic Resonance spectroscopy and Ultra-Performance Liquid Chromatography Mass Spectrometry. A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.

Prolonged Mechanical Circumferential Stretch Induces Metabolic Changes in Rat Inferior Vena Cava.

OBJECTIVE/BACKGROUND: Circumferential stretch on the vein wall has been suggested as a potential etiological factor in the development of varicose veins. However, the influence of vein wall stretch on vein metabolism has not yet been explored. The aim of this study was to investigate the effect of short and prolonged mechanical stretch on vein wall metabolism. METHODS: Circular segments of inferior vena cava from male Sprague-Dawley rats were exposed to normal 0.5-g (nonstretched) or high 2-g (stretched) tension for short (4 h) or prolonged (18 h) duration (five vein segments per group). Contraction response to phenylephrine (10-5 M) and KCl (96 mM) was elicited to observe the effect of circumferential stretch on vein function. The polar and organic metabolites in vein tissue were extracted using a bilayer extraction method. Aqueous and organic extracts were analyzed using nuclear magnetic resonance spectroscopy and ultra performance liquid chromatography coupled to mass spectrometry, respectively. Data acquired from both analytical platforms were analyzed using mathematical modeling. RESULTS: Increased concentrations of valine (p = .02) and choline (p = .03) metabolites and triglyceride moieties (p = .03) were observed in veins stretched for 18 h compared with the nonstretched/18 h group. DISCUSSION: Increased concentrations of branched chain amino acid valine and cell membrane constituent choline indicate increased muscle breakdown and increased metabolism of membrane phospholipids under stretch in an ex-vivo model. Increased intensities of triglyceride moieties in stretched vein segments for 18 h suggest that high pressure may induce an inflammatory response. CONCLUSION: This study has shown that prolonged mechanical circumferential stretch (18 h) alters the metabolic profile of rat inferior vena cava.

Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk: An Exploratory Study.

OBJECTIVE: Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. METHOD: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n = 5), and from asymptomatic patients (n = 5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. RESULTS: The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p = 10(-4)-10(-5)). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the ß-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. CONCLUSIONS: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and ß-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention.

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects.

Matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI-MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI-MSI; (3) To determine whether MALDI-MSI profiling of tumour-adjacent tissue can identify novel metabolic 'field effects' associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour-adjacent and tumour-remote healthy mucosa. We have referred to this 'field effect', exhibited by the tumour locale, as cancer-adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Clinical evaluation of microRNA expression profiling in non small cell lung cancer.

Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors. We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P = 0.010), miR-126* (P = 0.002), miR-30d (P = 0.012), miR-30e-5p (P < 0.001) and miR-451 (P < 0.001) were down-regulated, while miR-10a was not differentiated (P = 0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P = 0.022 and P = 0.037) and plasma (P = 0.045 and P = 0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P = 0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P = 0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC.

In-vitro identification of distinctive metabolic signatures of intact varicose vein tissue via magic angle spinning nuclear magnetic resonance spectroscopy.

OBJECTIVES: Nuclear magnetic resonance (NMR) spectroscopy is an established tool for metabolic profiling of tissues or biofluids with utility in identifying disease biomarkers and changes in enzymatic or gene expression. This pilot study aims to compare the metabolic profiles of intact varicose and non-varicose vein tissue via magic angle spinning (MAS) NMR spectroscopy with a view to promoting the understanding of the pathogenesis of varicose vein formation. METHODS: Varicose vein tissue (n = 8) was collected from patients undergoing varicose veins surgery. Control non-varicose great saphenous vein samples were collected from patients undergoing lower limb amputation (n = 3) and peripheral arterial bypass surgery (n = 5). Intact tissue samples (average weight 10.33 ± 0.8 mg) from each vein segment were analysed using 1D MAS (1)H NMR (600 MHz) spectroscopy. For selected vein samples, two-dimensional (2D) NMR experiments were performed. Differences between spectra from varicose and non-varicose tissues were elucidated using a variety of multivariate statistical analyses. RESULTS: The metabolic profiles of varicose veins samples were clearly differentiated from non-varicose veins samples. Lipid metabolites were present at a higher concentration in the non-varicose veins group whilst creatine, lactate and myo-inositol metabolites were more characteristic of the varicose veins group. CONCLUSION: We demonstrate differential metabolic profiles between varicose veins and non-varicose veins. Elucidating the metabolic signature underlying varicose veins can further improve our understanding of the biological mechanisms of disease initiation, progression, and aid in identifying putative therapeutic targets.