Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways

Rationale Nitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known. Objective Our objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity. Methods and measurements 34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay. Main results Of all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis. Conclusions In early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo

Activation and contraction of human ‘vascular’ smooth muscle cells grown from circulating blood progenitors

Blood outgrowth smooth muscle cells offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering and personalised medicine. However, little is known about these cells which has meant their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of blood outgrowth smooth muscle cells and vessel derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10 26 -6 M) increased cytosolic calcium in blood outgrowth smooth muscle cells fibroblasts. Increased calcium signal peaked between 10-20 seconds after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both blood outgrowth smooth muscle cells and vascular smooth muscle cells but not in fibroblasts. In summary, we show that functional responses of blood outgrowth smooth muscle cells are in line with vascular smooth muscle cells providing critical evidence of their application in biomedical research

Effect of COX-1 or COX-2 gene deletion on prostacyclin formation in the heart

Non peer reviewe

Relative expression of cyclo-oxygenase COX-1 and COX-2 in porcine endothelium subjected to chronic shear stress

Non peer reviewe

Effect of Type I–III IFNs on ET-1 release from HPASMCs.

<p>In the presence of TNFα, type I and type II, but not type III, interferons (IFNs) induce the release of ET-1 from human pulmonary artery smooth muscle cells (HPASMCs). Cells were treated with IFNs for 24 hours. Data are the mean ± standard error of the mean for n = 8. Within-group analysis was performed using one-way ANOVA followed by a Dunnett's post-test, where * indicates p<0.05 compared to control. Between-group analysis, for the effect of TNFα, was performed using two-way ANOVA followed by Bonferroni's post-test where # indicates p<0.05.</p

Effect of native versus pegylated IFN on IP10 release from HLMVECs.

<p>Effect of native IFNα2a or IFNα2b versus pegylated (PEG) forms on IP10 release from human lung microvascular endothelial cells (HLMVECs; A, B), human pulmonary artery smooth muscle cells (HPASMCs; C, D) and type II pneumocytes (A549 cells; E, F). Cells were treated with interferons (IFNs) alone (A, C and E) or in the presence of TNFα (10 ng/mL; B, D and F) for 24 hours. Data shown are mean ± standard error of the mean for n = 6 and were analysed using two-way ANOVA where * indicates p<0.05 for PEG-IFNα2a versus the other three IFNs.</p

Effect of PEG-IFN α2a isoforms on IP10 release from HLMVECs.

<p>Effect of different isoforms of PEG-IFN α2a on IP10 release from human lung microvascular endothelial cells (HLMVECs) co-treated with TNFα (10 ng/mL). Cells were treated for 24 hours with PEGIFNα2a or isoforms of PEGIFNα2a (K31, K134, K121) each at 10 ng/mL. Data are mean ± standard error of the mean for n = 7. Data have been normalized using IP10 release in the presence of 40KDa-PEGIFNα2a as 100%, and were analysed using a paired one sample t-test for each isoform or combination compared to PEGIFNα2a, where * indicates p<0.05. Intergroup analysis was performed using one-way ANOVA followed by Bonferroni's Multiple Comparison Test, where # indicates p<0.05.</p

Targeted metabolic profiling of amines and methylarginines in human plasma from healthy donors and patients with sepsis.

<p>Amine and methylarginine levels were measured using <b>(A)</b> UHPLC-MS/MS, LC-MS/MS and/or ELISA in the plasma of healthy donors and patients with sepsis at diagnosis (0 hours), 24 hours and 72 hours post diagnosis. Comparisons between levels of L-arginine from <b>(B)</b> UHPLC-MS/MS, LC-MS/MS and ELISA and <b>(C)</b> ADMA from LC-MS/MS and ELISA are shown at diagnosis (0 hours), 24 hours and 72 hours, post diagnosis. <b>(D)</b> Canonical pathways extracted from IPA software were based on an input of read outs comparing plasma levels of amines in healthy donors and patients with sepsis as a ratio. Data are ± SEM for n = 21 healthy donors and n = 38 patients with sepsis. Data was analysed by one-way ANOVA with Dunnett’s post-hoc test and Benjamini-Hochberg test with a false discovery rate of 0.05; *p<0.05.</p