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.