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Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeting and non-targeting gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.

Volatile compounds, abundant in breath, can be used to accurately diagnose and monitor a range of medical conditions. This offers a non-invasive, low-cost approach with screening applications; however, uptake of this diagnostic approach has been limited by conflicting published outcomes. Most published reports rely on large scale screening of the public, at single time points and without reference to ambient air. Here, we present a novel approach to volatile sampling from cellular headspace and mouse breath that incorporates multi-time point analysis and ambient air subtraction revealing compound flux as an effective proxy of active metabolism. This approach to investigating breath volatiles offers a new avenue for disease biomarker discovery and diagnosis. Using gas chromatography mass spectrometry (GC/MS), we focus on low molecular weight, metabolic substrate/by-product compounds and demonstrate that this non-invasive technique is sensitive (reproducible at ~1 µg cellular protein, or ~500,000 cells) and capable of precisely determining cell type, status and treatment. Isolated cellular models represent components of larger mammalian systems and we show that stress- and pathology-indicative compounds are detectable in mice, supporting further investigation using this methodology as a tool to identify volatile targets in human patients.

Volatile compounds contained in human breath reflect the inner workings of the body. A large number of studies have been published that link individual components of breath to disease, but diagnostic applications remain limited, in part due to inconsistent and conflicting identification of breath biomarkers. New approaches are therefore required to identify effective biomarker targets. Here, volatile organic compounds have been identified in the literature from four metabolically and physiologically distinct diseases and grouped into chemical functional groups (e.g. methylated hydrocarbons or aldehydes; based on known metabolic and enzymatic pathways) to support biomarker discovery and provide new insight on existing data. Using this functional grouping approach, principal component analysis doubled explanatory capacity from 19.1% to 38% relative to single individual compound approaches. Random forest and linear discriminant analysis reveal 93% classification accuracy for cancer. This review and meta-analysis provides insight for future research design by identifying volatile functional groups associated with disease. By incorporating our understanding of the complexities of the human body, along with accounting for variability in methodological and analytical approaches, this work demonstrates that a suite of targeted, functional volatile biomarkers, rather than individual biomarker compounds, will improve accuracy and success in diagnostic research and application.

Emerging studies from the ongoing covid-19 pandemic have implicated vascular dysfunction and endotheliitis in many patients presenting with severe disease. However, there is limited evidence for the expression of ACE2 (the principal co-receptor for Sars-Cov-2 cellular attachment) in vascular endothelial cells which has prompted the suggestion that the virus does not infect these cell types. However, the studies presented here demonstrate enhanced expression of ACE2 at the level of both mRNA and protein, in human pulmonary artery endothelial cells (PAECs) challenged with either IL-6 or hepcidin. Notably elevated levels both these iron-regulatory elements have been described in Covid-19 patients with severe disease and are further associated with morbidity and mortality. Additionally, levels of both IL-6 and hepcidin are often elevated in the elderly and in chronic disease settings, these populations being at greater risk of adverse outcomes from Sars-Cov-2 infection. A role for IL-6 and hepcidin as modulators of ACE2 expression seems plausible, additional, studies are required to determine if viral infection can be demonstrated in PAECs challenged with either of these iron-regulatory elements.

Introduction Pulmonary arterial hypertension (PAH) is characterised by vascular remodelling of pulmonary arterioles. Disrupted iron homeostasis linked to elevated hepcidin levels has been observed in PAH patients, and disruption of the hepcidin/ferroportin axis at the level of the pulmonary vasculature cells has been shown to contribute to proliferation of pulmonary artery smooth muscle cells. A role for Pulmonary artery endothelial cells (PAECs) linked to hepcidin has not been investigated. Objectives In this study we explored the influences of hepcidin-25 on PAEC gene expression targeting BMPRII, known to be dysfunctional in PAH. Methods Cells were challenged with Hepcidin-25 (1 μg/mL) or for comparison IL-6 (10 ng/mL). Transcriptional regulation was analysed by RT-PCR, protein expression by immunocytochemistry. Results Novel findings demonstrate that BMPRII mRNA expression is significantly down regulated in PAECs challenged with hepcidin-25 over a time course from 1 hour to 5 hours; figure 1 illustrates findings at 3 hours. IL-6 challenge was not able to replicate this response over the same time frame. In addition, Western blot analysis of cell lysates (n=2) showed an obvious loss of BMPRII protein expression in Hepcidin-25 challenged cells when compared to control and IL-6 challenged cell lysates. Conclusion This is the first report linking hepcidin-25 activity to potentially dysfunctional BMPRII responses in PAECs. Given the established role of hepcidin as regulator of cellular iron levels, a role for downstream signaling linked to iron accumulation in PAECs may offer a plausible mechanism for these observations and warrants further investigation. These studies may provide novel insights regarding emerging concepts of hepcidin driven proliferative and second messenger responses of relevance to PAH.

Vascular physiology predominates with endothelial cell-mediated smooth muscle responses to physiological conditions. Variations in cell-type responses are therefore important in vascular pathophysiology. Pulmonary Artery Hypertension (PAH) is characterised by vascular remodelling and proliferation of pulmonary artery smooth muscle cells (PASMC), increasing vascular resistance. Dysregulation of iron and elevated hepcidin are implicated in PAH and previously we have demonstrated hepcidin-mediated proliferation in PASMC. We hypothesise that responses to hepcidin induced iron overload are cell-type specific and we aim to comparatively analyse mitochondrial response in pulmonary arterial endothelial cells (PAEC) vs PASMC. Human PAEC or PASMC were treated with hepcidin (1 µg/ml) or IL-6 (10 ng/ml) for 24 hours. Mitochondria and mitochondrial Reactive Oxygen Species (mROS) were analysed with Mitotracker CMTMros and MitoSOX respectively, using confocal microscopy. Hepcidin treatment induced a significant reduction in fluorescence (***p =

Introduction Pulmonary arterial hypertension (PAH) is characterised by pulmonary vascular remodelling, increased vascular resistance and right ventricular failure. Dysregulation of iron homeostasis and increased proliferation in PAH have been linked to the iron-regulatory hormone hepcidin. Research from our lab has recently shown that hepcidin treatment in human pulmonary arterial smooth muscle cells (hPASMC) leads to an increased proliferative response possibly mediated by the haemoglobin (Hb) scavenger CD163. Objectives This study aims to characterise mitochondrial dysregulation in hPASMCs in relation to changes in iron homeostasis following treatment with hepcidin or haemoglobin. Methods hPASMCs were treated with hepcidin (1 µg/ml), IL-6 (10 ng/ml) and haemoglobin (3 µM) for 24 hours. Immunofluorescence of mitochondrial markers were analysed to assess changes to volume and intensity. Results Our findings indicate significant morphological changes with a 2 fold decrease in average mitochondrial volume following 24 hours of treatment with hepcidin or IL-6. In addition, haemoglobin treatment produced significant phenotypic changes with no significant change in volume or amount of mitochondria. Conclusion The findings presented here indicate that alterations in iron homeostasis influence global metabolism through mitochondrial dysfunction. This work presents novel insight into mitochondrial dynamics in relation to hepcidin driven proliferation in PAH.

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.