Disruption to endothelial cell homeostasis results in an extensive variety of human pathologies that are particularly relevant to major trauma. Circulating catecholamines, such as adrenaline and noradrenaline, activate endothelial adrenergic receptors triggering a potent response in endothelial function. The regulation of the endothelial cell metabolism is distinct and profoundly important to endothelium homeostasis. However, a precise catalogue of the metabolic alterations caused by sustained high catecholamine levels that results in endothelial dysfunction is still underexplored. Here, we uncover a set of up to 46 metabolites that exhibit a dose–response relationship to adrenaline-noradrenaline equimolar treatment. The identified metabolites align with the glutathione-ascorbate cycle and the nitric oxide biosynthesis pathway. Certain key metabolites, such as arginine and reduced glutathione, displayed a differential response to treatment in early (4 h) compared to late (24 h) stages of sustained stimulation, indicative of homeostatic metabolic feedback loops. Furthermore, we quantified an increase in the glucose consumption and aerobic respiration in endothelial cells upon catecholamine stimulation. Our results indicate that oxidative stress and nitric oxide metabolic pathways are downstream consequences of endothelial cell stimulation with sustained high levels of catecholamines. A precise understanding of the metabolic response in endothelial cells to pathological levels of catecholamines will facilitate the identification of more efficient clinical interventions in trauma patients.

Metabolic rewiring is one of the indispensable drivers of epithelial–mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP‐glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA‐mediated knock‐down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA‐mediated knock‐down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet‐derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA‐mediated knock‐down of PDGFRB downregulated UGDH expression, potentially via NFkB‐p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.

Background: In the year 2020 breast cancer was the most common form of cancer. Roughly 70% of breast cancers are estrogen receptor positive. MicroRNA-190b has previously been reported to be up-regulated in estrogen receptor positive breast cancers. Our group has previously demonstrated that microRNA-190b is hypomethylated in ER+ breast cancers, potentially leading to its upregulation. Results: In this study, using data from the Cancer Genome Atlas, we confirm that microRNA-190b is overexpressed in breast cancer via differential expression analysis and show that high expression of microRNA-190b results in more favorable outcomes in Luminal A patients (HR=0.29, 95% CI 0.12-0.71, P value=0.0063). MicroRNA190b target analysis, using immunoprecipitation of biotin labelled microRNA-190b, followed by RNA sequencing, identified RFWD3 as one of microRNA-190b’s regulatory targets in estrogen receptor positive breast cancer. Survival analysis of RFWD3 showed that elevated levels result in poorer overall survival in Luminal A breast cancer patients (HR = 2.22, 95% CI 1.33-3.71, P = 0.002). Gene ontology analysis of our sequencing results indicate that miR-190b may have a role in breast cancer development and/or tumorigenesis and that it may be a suitable tool in characterization between the estrogen receptor positive subtypes, Luminal A and Luminal B.Conclusions: We show that miR-190b targets RFWD3 in ER+ breast cancers leading to lower RFWD3 protein expression. Low levels of RFWD3 are associated with better outcomes in Luminal A breast cancer patients but not in Luminal B patients.

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