Background Pulmonary Arterial Hypertension (PAH) is a rare but fatal condition manifested by pulmonary vascular remodelling, increased pulmonary vascular resistance and right-heart failure. Disruption in iron handling and anaemia, caused by elevated iron-regulatory hormone hepcidin, is observed in PAH. Ferroportin the only known cellular iron-export protein is downregulated by hepcidin. As such, iron supplementation as a therapy is currently under clinical trial. However, it is also known that iron is both pro-oxidant and pro-proliferative. Latest evidence also points to sub-clinical haemolysis and the presence of free haemoglobin in PAH patients. We hypothesised that ferroportin would be expressed; be responsive to hepcidin challenge and have implications for the proliferation of human pulmonary artery smooth cells (hPASMCs).
Methods The mRNA levels of ferroportin was measured by RT-PCR, the protein expression was detected by western-blot analysis and quantified by ELISA. The sub-cellular distribution of ferroportin was visualised by immunocytochemistry (ICC). hPASMCs were pre-incubated with or without free haemoglobin and further challenged with increasing doses of hepcidin and the proliferative responses assessed by cyquant and/or BrdU incorporation assays. Some cells were also pre-incubated with LY2928057 (monoclonal antibody against ferroportin that stabilises cellular expression, Eli-Lily) in proliferation assays.
Results Basal ferroportin mRNA was detected in hPASMCs, but the mRNA levels were largely unaltered with hepcidin exposure (n = 3). A ~50KDa protein band representing ferroportin was detected under resting conditions while hepcidin challenge caused decrease in ferroportin protein levels (Figure 1). Basal ferroportin was uniformly distributed in the cells; however hepcidin treatment led to intense punctate/vesicular staining (n = 3). Finally, exposure to free haemoglobin alone or along with hepcidin increased proliferation of hPASMCs by 13.6% and 12.4% (p < 0.05, n = 3) respectively. Interestingly, pre-incubation of the cells with LY2928057 partly reversed this effect.
Conclusion This is the first report of ferroportin expression and regulation in hPASMCs. We suggest that targeting and manipulating the hepcidin-ferroportin axis using LY2928057 might prove a novel therapeutic approach for PAH.
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