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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.