Abstract 756: Genes Associated with Pulmonary Hypertension Regulate Osteoprotegerin in Human Pulmonary Artery Smooth Muscle Cells in vitro.
Pulmonary hypertension (PH) is a devastating and life threatening disease, characterised by narrowing and occlusion of small pulmonary arteries. Pulmonary artery smooth muscle cell (PA-SMC) migration and proliferation is thought to be the major contributing factor. Recent genetic information has highlighted the importance of both the bone morphogenetic protein receptor 2 (BMP-R2) and serotonin transporter (SERT) pathways. Despite this information, we are still unaware of how these genes interact with other associated pathways such as inflammation, to regulate disease. Osteoprotegerin (OPG) is a member of the TNF superfamily, which is expressed and secreted by a variety of cells including the vascular, immune and haematopoietic cells. OPG has an emerging role in vascular biology, and interestingly, BMPs, serotonin and IL-1 modulate OPG expression in other cell types. We hypothesised that OPG expression and secretion is regulated by BMP-R2 and/or SERT and inflammation, and that once secreted, OPG induces a pro-migratory and/or anti-apoptotic/pro-mitotic phenotype in PA-SMC, consistent with a role in the pathogenesis of PH. These studies used a combination of primary human PA-SMC, BMP-R2 siRNA, and stimulation with recombinant proteins. OPG gene expression was also studied in microarrays from PH patients carrying a BMP-R2 mutation. Secreted OPG was measured by ELISA; proliferation assessed by tritiated thymidine and migration by a transwell chamber assay. BMP-R2 siRNA increased OPG secretion 5-fold (n=4, p<0.05) compared to control oligos. BMP-2 (50 ng/ml) and TGF-B1 (1ng/ml) further increased OPG secretion. OPG gene expression was found to be 2-fold higher in PH patients with a BMP-R2 mutation, compared to control patients by microarray. Serotonin stimulated OPG release from human PA-SMC 2-fold compared to 0.1% FCS in a SERT-dependent manner (n=4, p<0.05) and IL-1 also increase OPG secretion 2-fold (n=4, p<0.05). Studies using recombinant OPG (50 ng/ml) showed it to be both pro-mitogenic (2-fold, n=4, p<0.05) and pro-migratory (3-fold, n=4, p<0.05) to PA-SMC in vitro. These data suggest that OPG may be a central molecule in the pathogenesis of pulmonary hypertension and provide a common link to the different pathways associated with disease.