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Background Pulmonary Arterial Hypertension (PAH) is a condition resulting from vascular remodelling and elevated pulmonary arterial pressure. Bone Morphogenetic Protein Receptor II (BMPR2) mutations have been strongly associated with heritable forms of PAH. Previous studies have demonstrated that the hepcidin/ferroportin axis acts to downregulate BMPR2 and promote proliferation in pulmonary artery smooth muscle. However, the role of hepcidin on pulmonary artery endothelial cells (PAEC) and BMPR2 downstream pathways is yet to be elucidated. Aims To investigate the influence of Hepcidin/IL-6 on PAEC proliferation, mitochondrial dysfunction, as well as BMPR2 and downstream pathway perturbation. Methods PAECs were treated with Hepcidin-25 (100 ng/mL or 1µg/mL) or Interleukin-6 (IL-6) (1 ng/mL or 10 ng/mL). Proliferation was determined using CyQuant assay, gene transcription was analysed using real-time PCR and protein expression by Western Blot (WB) and Enzyme-linked Immunosorbent Assay (ELISA). Results Hepcidin and IL-6 after 24h treatment cause PAECs to proliferate (n=4; p Conclusions These study findings uncover the complexity of the relationship between hepcidin and BMP-signalling in PAECs. Disruption in iron homeostasis and elevation in hepcidin levels have been reported in PAH populations, thus a role for a dysregulated hepcidin/ferroportin axis and downstream pathway disruption presents a potential mechanism for these observations. Nonetheless, further research is pivotal to fully elucidate the role of hepcidin in disrupting PAEC iron homeostasis and BMP-signalling.

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.

Background The link between pulmonary arterial hypertension (PAH) and haemolytic anaemias, such as sickle-cell disease and thalassaemia, is well established. Recent studies have implicated sub-clinical haemolysis and the release of cell free haemoglobin (CFH) in idiopathic PAH. The interaction between CFH and pulmonary artery endothelial cells (PAECs) could induce endothelial dysfunction, a key component of the pathophysiology of PAH. Objectives This study aims to investigate the role of CFH in PAEC dysfunction, defined in terms of intracellular and mitochondrial reactive oxygen species (ROS) generation, altered cell proliferation indices and changes in gene transcription of the ROS-generating enzyme NADPH oxidase-2 (Nox2). Methods Cultured human PAECs (hPAECs) were challenged with 10 μM haemoglobin (Hb) or no treatment (control) for 24 hours. Flow cytometry was used to measure total intracellular ROS (dihydroethidium assay), mitochondrial ROS (MitoSOX assay) and cell cycle profile using propidium iodide. Nox2 gene expression was measured using RT-qPCR. Cell proliferation was measured using the BrdU assay. Results Total intracellular and mitochondrial ROS production in HPAECs increased (∼3-fold) following Hb challenge compared to control. Additionally, Nox2 mRNA expression was greater in hPAECs treated with Hb for durations of 1 or 2 hours compared to control. Hb-treated hPAECs displayed a significant decrease (*p Conclusions These findings suggest that hPAECs exposed to Hb undergo an increase in intracellular and mitochondrial ROS production, which is also associated with an upregulation in Nox2 gene expression. Results from the cell cycle and BrdU assays suggest contrasting proliferative responses to Hb exposure, but warrant further investigation into possible changes in apoptotic or cell repair processes. Further studies are warranted to investigate the role of these processes in the PAH disease setting.

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.

Pulmonary arterial hypertension (PAH) is characterised by vascular remodelling of pulmonary arterioles. Disrupted iron homeostasis and elevated hepcidin are implicated in PAH, although exact mechanisms remain unknown. Hepcidin targets ferroportin, the only known iron exporter to cause its internalisation. Earlier we established the expression of ferroportin in human pulmonary artery smooth muscle cells (hPASMCs) and also demonstrated its enhanced proliferation and migration when exposed to hepcidin conditioned media (HCM) from pulmonary artery endothelial cells (hPAECs). In this study, we further explored the dose dependent effect of hepcidin in the CM, its role in ferroportin expression and the ability of LY2928057-mAb against ferroportin in reversing responses elicited by HCM. hPAECs were challenged with hepcidin (0.1-1ug/mL). Proliferation and migration were analysed using xCELLigence RTCA by measuring the changes in cell index. Ferroportin expression was analysed by immunocytochemistry; GDF-15 and IREB2 were quantified by ELISA. In the absence of hPAECs, HCM had little or no effect on either proliferation or migration of hPASMCs. Confocal imaging revealed decreased ferroportin expression in hPASMC treated with HCM from hPAECs compared to HCM without hPAECs. LY2928057, inhibited proliferation induced by HCM and also decreased IREB2 levels in hPASMCs. LY2928057 also increased GDF-15 expression in hepcidin-treated hPAECs. Together these data suggest a vital role for the hepcidin-ferroportin axes in controlling proliferation, migration and release of mediators by pulmonary vascular cells, relevant to PAH and that LY2928057 could potentially reverse some these effects