A bioassay system of autologous human endothelial, smooth muscle cells and leucocytes for use in drug discovery, phenotyping and tissue engineering

Purpose: Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leucocytes to generate a same donor ‘vessel in a dish’ bioassay. Basic procedures: Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs) and leucocytes were obtained from 4 donors. Cells were treated in mono and cumulative co-culture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumour necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines. Main findings: Co-cultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels. Principle conclusions: For the first time, we report a proof of concept study where autologous blood outgrowth ‘vascular’ cells and leucocytes were studied alone and in co-culture. This novel bioassay has utility in vascular biology research, patient phenotyping, drug testing and tissue engineering

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease

Plasma S100A8/A9 heterodimer is an early prognostic marker of acute kidney injury associated with cardiac surgery

We investigated whether plasma levels of the inflammation marker S100A8/A9, could predict acute kidney injury (AKI) onset in patients undergoing cardiac surgery necessitating cardiopulmonary bypass (CPB). Patients & methods: Plasma levels of S100A8/A9 and other neutrophil cytosolic proteins were measured in 39 patients pre- and immediately post-CPB. Results: All markers increased significantly post-CPB with S100A8/A9, S100A12 and myeloperoxidase levels significantly higher in patients who developed AKI within 7 days. S100A8/A9 had good prognostic utility for AKI, with an area under the receiver operating characteristic curve of 0.81 (95% CI: 0.676–0.949) and a cut-off value of 10.6 μg/ml (85.7% sensitivity and 75% specificity) irrespective of age. Conclusion: Plasma S100A8/A9 levels immediately after cardiac surgery, can predict onset of AKI, irrespective of age

Somatic variants in epigenetic modifiers can predict failure of response to imatinib but not to second-generation tyrosine kinase inhibitors

There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukaemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase-inhibitor. Our inability to predict these 'high-risk' individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase-inhibitors and probability of disease progression. 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukaemia began with imatinib (n=62) or second-generation tyrosine kinase-inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase-inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic-myeloid-leukaemia-related survival in the imatinib but not the second-generation tyrosine kinase-inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukaemia variants suggest a multi-step development of chronic myeloid leukaemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase-inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression

Pericyte degeneration causes white matter dysfunction in the mouse central nervous system.

Diffuse white-matter disease associated with small-vessel disease and dementia is prevalent in the elderly. The biological mechanisms, however, remain elusive. Using pericyte-deficient mice, magnetic resonance imaging, viral-based tract-tracing, and behavior and tissue analysis, we found that pericyte degeneration disrupted white-matter microcirculation, resulting in an accumulation of toxic blood-derived fibrin(ogen) deposits and blood-flow reductions, which triggered a loss of myelin, axons and oligodendrocytes. This disrupted brain circuits, leading to white-matter functional deficits before neuronal loss occurs. Fibrinogen and fibrin fibrils initiated autophagy-dependent cell death in oligodendrocyte and pericyte cultures, whereas pharmacological and genetic manipulations of systemic fibrinogen levels in pericyte-deficient, but not control mice, influenced the degree of white-matter fibrin(ogen) deposition, pericyte degeneration, vascular pathology and white-matter changes. Thus, our data indicate that pericytes control white-matter structure and function, which has implications for the pathogenesis and treatment of human white-matter disease associated with small-vessel disease