Introduction and objectives Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive pulmonary vascular remodelling, a key component of which is the proliferation and migration of pulmonary arterial smooth muscle cells (PA-SMCs). Although current therapies are good at alleviating symptoms, they do not reverse the underlying pulmonary vascular remodelling. We have previously demonstrated that the secreted glycoprotein, osteoprotegerin (OPG, TNFRSF11B), is elevated within pulmonary vascular lesions and serum from idiopathic PAH (IPAH) patients and induces PA-SMC proliferation and migration in vitro. Furthermore, genetic deletion or antibody blockade of OPG can prevent and reverse disease in pre-clinical animal models. However, how OPG signals to mediate PA-SMC phenotype remains unclear. We therefore aimed to characterise the OPG signalling cascade in PA-SMCs, and identify the receptor through which this is mediated.
Methods PA-SMCs were stimulated with 0.2% FCS and OPG (50 ng/ml) for 10 and 60 min. Phosphorylation targets were identified by Kinex antibody microarray (Kinexus, Canada). An RNA expression microarray (Agilent) was performed on PA-SMCs following 6-hour OPG stimulation. OPG binding partners were identified following reverse transfection of HEK293 cells with 2054 human membrane proteins (Retrogenix, Sheffield, UK). Interactions were confirmed in PA-SMCs by co-immunoprecipitation. PA-SMCs were pre-treated with Fas neutralising antibody (1500 ng/ml), TRAIL antibody (1500 ng/ml) or both antibodies, 30 min before OPG stimulation. Proliferation was assessed after 72 h.
Results OPG induced significant activation of CDK4 and CDK5, HSP27 and ERK1/2, and significant decrease in phospho-mTOR. OPG significantly altered the expression of 57 PAH-associated genes, including TRAIL. Four novel OPG interactions with IL1RAcP, Fas, TMPRSS11D and GAP43 were identified and we confirmed OPG interaction with IL1RAcP and Fas in PA-SMC. Fas RNA expression was elevated in IPAH PA-SMCs and protein expression was elevated in the right ventricle and pulmonary artery from IPAH patients. Fas blockade reduced OPG-induced proliferation by ~40%, which was further reduced by simultaneous TRAIL blockade. Fas blockade also prevented OPG-induced PDGFRA and TNC RNA expression.
Conclusions These studies begin to reveal the intracellular signalling mechanisms and receptor through which OPG induces PA-SMC proliferation, further highlighting the therapeutic potential of targeting OPG in PAH.