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S97 Haemoglobin challenge induces dysfunction in human pulmonary artery endothelial cells: potential relevance to pulmonary artery hypertension
  1. MS Bukhari1,
  2. M Mohd-Ghazaly2,
  3. QK Toe1,
  4. GJ Quinlan1,
  5. SJW Wort1
  1. 1Imperial College London, London, UK
  2. 2Universiti Malaysia Terengganu, Kuala Nerus, Malaysia

Abstract

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<0.05) in the percentage of cells in S and G2/M phases compared to control (see figure). In contrast, the BrdU results indicated a significant increase (∼1.8 fold) in proliferation in response to Hb treatment (**p<0.005).

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.

Abstract S97 Figure 1

Change in cell proliferation

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