Evidence of a role for osteoprotegerin in the pathogenesis of pulmonary arterial hypertension - PubMed (original) (raw)
Evidence of a role for osteoprotegerin in the pathogenesis of pulmonary arterial hypertension
Allan Lawrie et al. Am J Pathol. 2008 Jan.
Abstract
Pulmonary artery smooth muscle cell (PA-SMC) migration and proliferation are key processes in the pathogenesis of pulmonary arterial hypertension (PAH). Recent information suggests that abnormalities in the bone morphogenetic protein (BMP) receptor 2 (BMP-R2) signaling pathway are important in PAH pathogenesis. It remains unclear whether and how this pathway interacts with, for example, serotonin (5-HT) and inflammation to trigger and/or sustain the development of PAH. The secreted glycoprotein osteoprotegerin (OPG) is emerging as an important regulatory molecule in vascular biology and is modulated by BMPs, 5-HT, and interleukin-1 in other cell types. However, whether OPG is expressed by PA-SMCs within PAH lesions and plays a role in PAH is unknown. Immunohistochemistry of human PAH lesions demonstrated increased OPG expression, and OPG was significantly increased in idiopathic PAH patient serum. Recombinant OPG stimulated proliferation and migration of PA-SMCs in vitro, and BMP-R2 RNA interference increased OPG secretion. Additionally, both 5-HT and interleukin-1 also increased OPG secretion. These data are the first to demonstrate that OPG is increased in PAH and that it can regulate PA-SMC proliferation and migration. OPG may provide a common link between the different pathways associated with the disease, potentially playing an important role in the pathogenesis of PAH.
Figures
Figure 1
OPG expression localizes to medial areas of IPAH lesions. Lung sections were immunohistochemically stained for OPG (A–C), TRAIL (D–F), RANKL (G–I), CD31 (J–L), smooth muscle actin (SMA; M–O), CD68 (P–R), and mouse IgG isotype control (S–U). A, D, G, J, M, P, and S show serial sections of a control donor pulmonary artery. B, E, H, K, N, Q, and T show staining of serial sections of a concentric lesion and C, F, I, L, O, R, and U, of a plexiform IPAH lesions. Magnification, ×400. Scale bar = 50 μm (A).
Figure 2
OPG expression is increased in IPAH patient serum. Serum was collected from IPAH and control patients and analyzed for OPG concentration by ELISA. Each spot represents an individual patient, and the horizontal line marks the mean, n = 33 controls and 38 IPH samples.
Figure 3
Recombinant OPG induces proliferation and migration of human PA-SMCs. Human PA-SMCs were serum starved for 48 hours before stimulation with recombinant OPG or 10 ng/ml PDGF-BB. A: Proliferation was assessed at 36 hours by tritiated thymidine incorporation and expressed as fold increase over unstimulated cells (ctrl). B: Migration was measured at 6 hours using a Boyden Chamber assay and normalized relative to unstimulated cells (ctrl). Bars represent mean ± SEM from four different experiments; **P < 0.001, *P < 0.05 compared with 0.1% fetal calf serum-treated control cells (ctrl).
Figure 4
Reduced BMP-R2 expression increased OPG secretion. Human PA-SMCs were either mock transfected (Ctrl) or transfected with 100 nmol/L Cyclophilin B siRNA (CycloB si) or BMP-R2 siRNA (BMP-R2 si) oligos. A: RNA was collected 72 hours after transfection, assayed for BMP-R2 gene expression by TaqMan PCR, and normalized to expression of β-2-microglobulin (B2M). BMP-R2 siRNA significantly reduced BMP-R2 gene expression 80% compared with mock-transfected cells. B: Conditioned media was collected 72 hours after treatment, assayed for OPG via ELISA, and normalized for effects on cell number by Coulter Counting. Bars represent mean ± SEM from four replicate experiments. *P < 0.05 versus nontreated control cells.
Figure 5
5-HT stimulation increases OPG secretion in a SERT-dependent manner. A: Human PA-SMCs were serum starved for 48 hours before stimulation with increasing concentrations of 5-HT (0.3 to 3 μmol/L). Conditioned medium was collected 72 hours after treatment, assayed for OPG via ELISA, and normalized for effects on cell number by Coulter Counting. Maximal stimulation was observed at 1 μmol/L 5-HT. B: To determine the effect of SERT, the cells were treated with 5 μmol/L fluoxetine, a SERT inhibitor, or with dimethyl sulfoxide (vehicle) alone before stimulation with 1 μmol/L 5-HT. Bars represent mean ± SEM from four replicate experiments. *P < 0.05 versus nontreated control cells.
Figure 6
IL-1 signaling stimulates OPG secretion. Human PA-SMCs were serum starved for 48 hours before stimulation with IL-1α (0.1 to 3 ng/ml) (A) or IL-1β (3 to 30 ng/ml) (B). Conditioned media were collected 72 hours after treatment, assayed for OPG via ELISA, and normalized for effects on cell number by Coulter Counting. Maximal OPG secretion was observed at 1 ng/ml IL-1α and 10 ng/ml IL-1β. Bars represent mean ± SEM from three replicate experiments. *P < 0.05 versus nontreated control cells.
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