FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2040642173> ?p ?o ?g. }

• W2040642173 endingPage "264" @default.
• W2040642173 startingPage "256" @default.
• W2040642173 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. 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. Abnormal proliferation and migration of pulmonary artery smooth muscle cells (PA-SMCs) play a central role in the pathogenesis of pulmonary arterial hypertension (PAH).1Pietra GG Edwards WD Kay JM Rich S Kernis J Schloo B Ayres SM Bergofsky EH Brundage BH Detre KM Histopathology of primary pulmonary hypertension: a qualitative and quantitative study of pulmonary blood vessels from 58 patients in the National Heart, Lung, and Blood Institute, Primary Pulmonary Hypertension Registry.Circulation. 1989; 80: 1198-1206Crossref PubMed Scopus (401) Google Scholar The association between cases of idiopathic PAH (IPAH) and heterozygous germline mutations in the gene encoding bone morphogenetic protein receptor type 2 (BMP-R2)2Lane K Machado R Pauciulo M Thomson J Philips III, JA Loyd JE Nichols W Trembath R Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension.Nat Genet. 2000; 26: 81-84Crossref PubMed Scopus (1208) Google Scholar or a polymorphism in the serotonin [5-hydroxytryptamine (5-HT)] transporter (SERT)3Eddahibi S Humbert M Fadel E Raffestin B Darmon M Capron F Simonneau G Dartevelle P Hamon M Adnot S Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension.J Clin Invest. 2001; 108: 1141-1150Crossref PubMed Scopus (488) Google Scholar have identified candidate pathways that may be involved in disease pathogenesis. Only a subset of patients with either genotype develop IPAH, and secondary pulmonary arterial hypertension affects only a minority of patients with associated primary illnesses such as systemic sclerosis. This suggests that multiple “hits” involving several cellular signaling pathways are required to produce the dysregulated PA-SMC proliferation and migration seen in PAH. The medial hypertrophy, intimal fibrosis, and in some cases, plexiform lesions of PAH are often accompanied by extensive perivascular inflammation.4Ziesche R Petkov V Williams J Zakeri SM Mosgoller W Knofler M Block LH Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue.Proc Natl Acad Sci USA. 1996; 93: 12478-12483Crossref PubMed Scopus (67) Google Scholar Moreover, the levels of several inflammatory mediators, including interleukin (IL)-1 and IL-6, are elevated in patients with pulmonary hypertension.5Humbert M Monti G Brenot F Sitbon O Portier A Grangeot-Keros L Duroux P Galanaud P Simonneau G Emilie D Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension.Am J Respir Crit Care Med. 1995; 151: 1628-1631Crossref PubMed Scopus (634) Google Scholar These observations suggest an inflammatory component to the underlying mechanism of disease and that inflammatory cytokines may interact with dysregulated BMP and/or 5-HT signaling to create the multiple hits required for the development of PAH. However, the identity of key downstream molecules remains unknown.Osteoprotegerin (OPG, TNFRSF11B), a member of the tumor necrosis factor receptor superfamily, is a secreted basic glycoprotein that exists as a 60-kDa monomer and a 120-kDa disulfide-linked homodimer.6Yamaguchi K Kinosaki M Goto M Kobayashi F Tsuda E Morinaga T Higashio K Characterization of structural domains of human osteoclastogenesis inhibitory factor.J Biol Chem. 1998; 273: 5117-5123Crossref PubMed Scopus (185) Google Scholar OPG is expressed and secreted by a variety of tissues, including the heart and lung,7Simonet WS Lacey DL Dunstan CR Kelley M Chang MS Luthy R Nguyen HQ Wooden S Bennett L Boone T Shimamoto G DeRose M Elliott R Colombero A Tan HL Trail G Sullivan J Davy E Bucay N Renshaw-Gegg L Hughes TM Hill D Pattison W Campbell P Sander S Van G Tarpley J Derby P Lee R Boyle WJ Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4299) Google Scholar and has been primarily studied in its role of regulating bone turnover and apoptosis in health and in disease.8Holen I Shipman CM Role of osteoprotegerin (OPG) in cancer.Clin Sci (Lond). 2006; 110: 279-291Crossref PubMed Scopus (94) Google Scholar, 9Roodman GD Regulation of osteoclast differentiation.Ann NY Acad Sci. 2006; 1068: 100-109Crossref PubMed Scopus (155) Google Scholar In these situations, OPG functions as a secreted decoy receptor, competing either with receptor activator of nuclear factor-κB (RANK) for the binding of RANK ligand (RANKL) to regulate osteoclast differentiation and activation7Simonet WS Lacey DL Dunstan CR Kelley M Chang MS Luthy R Nguyen HQ Wooden S Bennett L Boone T Shimamoto G DeRose M Elliott R Colombero A Tan HL Trail G Sullivan J Davy E Bucay N Renshaw-Gegg L Hughes TM Hill D Pattison W Campbell P Sander S Van G Tarpley J Derby P Lee R Boyle WJ Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4299) Google Scholar or with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), preventing binding to membrane-associated death receptors (DR4 and DR5) to trigger apoptosis of multiple cell types.10Emery JG McDonnell P Burke MB Deen KC Lyn S Silverman C Dul E Appelbaum ER Eichman C DiPrinzio R Dodds RA James IE Rosenberg M Lee JC Young PR Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL.J Biol Chem. 1998; 273: 14363-14367Crossref PubMed Scopus (1049) Google ScholarThere is increasing evidence that OPG may play an important role in vascular disease. OPG knockout mice exhibit increased vascular calcification,11Bucay N Sarosi I Dunstan CR Morony S Tarpley J Capparelli C Scully S Tan HL Xu W Lacey DL Boyle WJ Simonet WS Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2111) Google Scholar and OPG concentrations are elevated in plasma of patients with peripheral and coronary artery disease and systemic hypertension.12Avignon A Sultan A Piot C Elaerts S Cristol JP Dupuy AM Osteoprotegerin is associated with silent coronary artery disease in high-risk but asymptomatic type 2 diabetic patients.Diabetes Care. 2005; 28: 2176-2180Crossref PubMed Scopus (94) Google Scholar, 13Ziegler S Kudlacek S Luger A Minar E Osteoprotegerin plasma concentrations correlate with severity of peripheral artery disease.Atherosclerosis. 2005; 182: 175-180Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 14Rasmussen LM Tarnow L Hansen TK Parving HH Flyvbjerg A Plasma osteoprotegerin levels are associated with glycaemic status, systolic blood pressure, kidney function and cardiovascular morbidity in type 1 diabetic patients.Eur J Endocrinol. 2006; 154: 75-81Crossref PubMed Scopus (137) Google Scholar Additionally, OPG has recently been shown to contribute to plaque progression in an atherosclerotic ApoE−/− mouse model.15Bennett BJ Scatena M Kirk EA Rattazzi M Varon RM Averill M Schwartz SM Giachelli CM Rosenfeld ME Osteoprotegerin inactivation accelerates advanced atherosclerotic lesion progression and calcification in older ApoE−/− mice.Arterioscler Thromb Vasc Biol. 2006; 26: 2117-2124Crossref PubMed Scopus (253) Google Scholar In the context of a potential link between OPG and PAH, BMPs, 5-HT, and the inflammatory cytokine IL-1 are all known to up-regulate OPG in a variety of cell types.16Hofbauer LC Dunstan CR Spelsberg TC Riggs BL Khosla S Osteoprotegerin production by human osteoblast lineage cells is stimulated by vitamin D, bone morphogenetic protein-2, and cytokines.Biochem Biophys Res Commun. 1998; 250: 776-781Crossref PubMed Scopus (274) Google Scholar, 17Brändström H Bjorkman T Ljunggren O Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells.Biochem Biophys Res Commun. 2001; 280: 831-835Crossref PubMed Scopus (93) Google Scholar, 18Gustafsson BI Thommesen L Stunes AK Tommeras K Westbroek I Waldum HL Slordahl K Tamburstuen MV Reseland JE Syversen U Serotonin and fluoxetine modulate bone cell function in vitro.J Cell Biochem. 2006; 98: 139-151Crossref PubMed Scopus (132) Google Scholar Taken together, these observations suggest that OPG may play an important role in the control of vascular cell behavior, regulated at least in part by signaling pathways implicated in PAH pathogenesis. Because the role of OPG signaling in PAH has not previously been studied, we hypothesized that these signaling pathways could converge to dysregulate expression of OPG and that this in turn would lead to increased PA-SMC proliferation and migration. We present data here to support this hypothesis that demonstrates for the first time that abnormalities in BMP, 5-HT, and inflammatory signaling result in heightened expression of OPG in PA-SMCs. Additionally, we show that OPG increases PA-SMC proliferation and migration, indicating the potential to contribute to the pathogenesis of PAH.Materials and MethodsLung and Serum Samples from Patients with IPAHLung tissue samples (n = 12, 4 male and 8 female; mean age, 40 ± 11 years) from patients with familial IPAH associated with mutant BMPR2 (n = 6), IPAH without BMPR2 mutation (n = 6), and control subjects undergoing lung resection for cancer or from unused donor-lung (n = 6, 4 male and 2 female; mean age, 55 ± 21 years) were obtained from the Papworth Hospital NHS Trust tissue bank as previously described.19Yang X Long L Southwood M Rudarakanchana N Upton PD Jeffery TK Atkinson C Chen H Trembath RC Morrell NW Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension.Circ Res. 2005; 96: 1053-1063Crossref PubMed Scopus (287) Google Scholar Serum was also obtained from 38 patients with IPAH (7 with, 13 without, and 18 not genotyped for BMP-R2 mutations; 10 male and 28 female; mean age, 44 ± 14 years) and from a control group comprising healthy adult volunteers and patients from an elective orthopedic preadmission clinic (n = 33, 20 male and 13 female; mean age, 58 ± 16 years). All subjects gave informed written consent, and the study was approved by the Local Research Ethics Committee.ImmunohistochemistryFormalin-fixed, paraffin-embedded lung samples were serially sectioned at 5 μm and processed using 0.6 mol/L sodium citrate buffer (pH 6) antigen retrieval where required, as previously described.19Yang X Long L Southwood M Rudarakanchana N Upton PD Jeffery TK Atkinson C Chen H Trembath RC Morrell NW Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension.Circ Res. 2005; 96: 1053-1063Crossref PubMed Scopus (287) Google Scholar Serial sections were stained with goat anti-human osteoprotegerin (AF805; R&D Systems, Abingdon, Oxfordshire, UK), mouse monoclonal anti-human TRAIL (Vector Laboratories, Peterborough, UK), or mouse monoclonal anti-human RANKL (MAB626; R&D Systems). To colocalize components to the vascular endothelium and SMCs, immunohistochemical markers against endothelial cells (anti-CD31; DakoCytomation, Ely, Cambridgeshire, UK) and SMCs (anti-α-smooth muscle actin; DakoCytomation) were also used. Antibodies were incubated for 1 hour and labeled using the relevant streptavidin ABC peroxidase technique [DakoCytomation or Vector Laboratories (for OPG as only goat primary)] and visualized with 3,3′-diaminobenzidine (DakoCytomation).OPG Enzyme-Linked Immunosorbent AssayOPG concentration in cell culture medium and patient serum was measured by enzyme-linked immunosorbent assay (ELISA) as previously described.20Holen I Croucher PI Hamdy FC Eaton CL Osteoprotegerin (OPG) is a survival factor for human prostate cancer cells.Cancer Res. 2002; 62: 1619-1623PubMed Google Scholar For in vitro studies on cell culture medium, OPG was normalized to picograms of OPG per cell by performing cell counts on the monolayer using a Coulter counter.Cell Culture and Phenotypic AssaysHuman PA-SMCs were purchased from Cascade Biologics (Mansfield, UK) and maintained as previously described.21Lawrie A Spiekerkoetter E Martinez EC Ambartsumian N Sheward WJ MacLean MR Harmar AJ Schmidt AM Lukanidin E Rabinovitch M Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease.Circ Res. 2005; 97: 227-235Crossref PubMed Scopus (129) Google Scholar Before stimulation, PA-SMCs were synchronized by incubating in Dulbecco's modified Eagle's medium containing PSA Solution (Cascade Biologics) and 0.2% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA) for 48 hours. Cell proliferation was assessed using tritiated thymidine incorporation and Coulter Counting, and migration was assessed using a Boyden chamber assay as previously described.22Leung WC Lawrie A Demaries S Massaeli H Burry A Yablonsky S Sarjeant JM Fera E Rassart E Pickering JG Rabinovitch M Apolipoprotein D and platelet-derived growth factor-BB synergism mediates vascular smooth muscle cell migration.Circ Res. 2004; 95: 179-186Crossref PubMed Scopus (27) Google Scholar Where required, cells were stimulated with recombinant human OPG (R&D Systems), recombinant human IL-1β (R&D Systems), or serotonin creatinine sulfate complex (Sigma, St. Louis, MO) at the stated concentrations. Pre-incubation with the SERT inhibitor fluoxetine (Sigma) was performed as previously described.21Lawrie A Spiekerkoetter E Martinez EC Ambartsumian N Sheward WJ MacLean MR Harmar AJ Schmidt AM Lukanidin E Rabinovitch M Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease.Circ Res. 2005; 97: 227-235Crossref PubMed Scopus (129) Google ScholarShort Interfering RNA TransfectionsRNA interference was induced by transient transfection of 100 nmol/L short interfering RNA (siRNA) oligonucleotides targeting either BMP-R2 or cyclophilin B (Dharmacon, Lafayette, CO) complexed with DharmaFECT2 lipid transfection reagent (Dharmacon) according to the manufacturer's instructions. Knockdown of BMP-R2 and was confirmed by TaqMan Gene Expression Assay (Assay ID Hs00176148_m1; Applied Biosystems, Foster City, CA) at 72 hours after transfection.Statistical AnalysesStatistical analysis was performed using either a Mann-Whitney U-test when comparing two groups or a repeated measure analysis of variance followed by the Newman-Keuls post hoc test with a 95% confidence level. P < 0.05 was deemed statistically significant (Prism 4.0c for Macintosh; GraphPad, San Diego, CA).ResultsOPG Expression Is Increased in Concentric and Plexiform IPAH LesionsImmunohistochemical analysis of human lung tissue samples demonstrated a very low level of OPG expression in sections of control (transplant donor) lung, localized mainly in a few lumenal endothelial cells (Figure 1A). In the IPAH lung sections, diffuse medial staining and strong cellular immunoreactivity was observed within both concentric (Figure 1B) and plexiform lesions (Figure 1C) of remodeled pulmonary arteries. Further immunohistochemical analysis was performed to determine the expression patterns of OPG-binding partners, TRAIL, RANKL, and Syndecan-1. Weak immunoreactivity for TRAIL was observed within the lumenal endothelial cells of control vessels (Figure 1D), whereas increased staining was observed in the outer medial layers and lumenal endothelium of the concentric IPAH lesions (Figure 1E) and throughout the plexiform lesions (Figure 1F). RANKL expression was barely detectable in the control lung (Figure 1G). In contrast, diffuse immunoreactivity was observed around the luminal edge and the outermost layers of the concentric lesions (Figure 1H). Plexiform lesions showed diffuse immunoreactivity for RANKL throughout the lesions (Figure 1I). Syndecan-1 was not detected in either control or IPAH sections (data not shown). To characterize the cellular composition of the lesions in relation to the expression of these proteins, serial sections were stained for cell-specific markers. CD31 was used to identify endothelial cells (Figure 1, J–L), smooth muscle actin for smooth muscle cells (Figure 1, M–O), and CD68 to detect monocyte/macrophage infiltration (Figure 1, P–R). Staining of control donor lung sections demonstrates a typical thin-walled artery comprising smooth muscle cells, intact endothelium, and small numbers of CD68+ monocytes/macrophages (Figure 1, J, M, and P). In contrast, the concentric lesions show characteristic medial thickening due to increased SMCs (Figure 1N) and the consequent reduced lumen with an intact endothelium (Figure 1K); a slight increase in CD68+ staining was observed around the artery (Figure 1Q). The medium was shown to consist of primarily SMCs, indicating that the medial expression of OPG was by SMCs. The expression of RANKL and TRAIL appeared to be associated with both endothelium and SMCs of concentric lesions. The plexiform lesions show an increased CD31 staining (Figure 1L) and disorganized areas of smooth muscle actin+ cells (Figure 1O) within the areas bordered by CD31 staining. A few CD68+ cells were also observed, mainly in the interstitial regions of the lung (Figure 1R). The localization of OPG, TRAIL, and RANKL appears similar to that within concentric lesions.OPG Expression Is Increased in IPAH Patient SerumGiven that OPG is a secreted molecule and that increased immunoreactivity was observed within IPAH lesions, we next determined OPG levels in patient sera. OPG levels in sera from IPAH patients was significantly elevated by more than threefold (1530 ± 191.3 pg/ml, n = 33 control patients versus 4710 ± 369.2 pg/ml, n = 38 IPAH patients; P < 0.0001) compared with unaffected control subjects (Figure 2). A summary of the patient demographics, including World Health Organization functional class, BMP-R2 mutation status, and treatment, is shown in Table 1. A breakdown of each individual IPAH patient OPG levels, treatment, and hemodynamic profile is shown in Table 2. No correlation was found between OPG concentration and World Health Organization class or hemodynamic measurements, although group numbers are small. Further studies on larger patient groups will be required to provide definitive information on these aspects. Further subanalysis also revealed no significant difference in the serum concentration of OPG between those IPAH patients who carried a BMP-R2 mutation compared with those without (4537 ± 619 pg/ml, n = 7 BMP-R2 mutation versus 5595 ± 526.6 pg/ml, n = 13 BMP-R2 normal; 18 patients were of unknown BMP-R2 status, 4138 ± 615.6 pg/ml).Figure 2OPG 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Total Population Demographics, WHO Class, BMP-R2 Status, and TreatmentsControls [n (%)]IPAH patients [n (%)]n3338Age at sample collection (years ± SD)57.6 ± 15.945.3 ± 14.5Sex Male20 (60.6)10 (26.3) Female13 (39.4)28 (73.7)WHO functional class0 (0) I13 (34.2) II21 (55.3) III4 (10.5) IVBMP-R2 mutation status Positive7 (18.4) Negative13 (34.2) Unknown18 (47.4)Targeted treatment None8 (21.1) Bosentan9 (23.7) Epoprostenol7 (18.4) Iloprost (intravenous)2 (5.3) Iloprost (nebulized)2 (5.3) Sildenafil1 (2.6) Combined and miscellaneous8 (21.1)Osteoprotegerin (pg/ml) (mean ± SEM)1530 ± 191.34710 ± 369.2WHO, World Health Organization. Open table in a new tab Table 2Individual IPAH Patient Demographic Profile and OPG ExpressionIDBMP-R2 mutationSexDisease duration*Denotes time from first presentation to sample time in years.WHO†World Health Organization (WHO) Functional Class for Pulmonary Arterial Hypertension.;Rx‡Treatments for PAH were Epoprostenol (E), intravenous Iloprost (IV I), nebulized Iloprost (Neb I), Bosentan (B), and Sildenafil (S).RAPmPAPCOCITPROPG29−F6.6IINoneN/AN/AN/AN/AN/A6043.035−M3.5IIINeb I10554.9211.26191.739−F6.7IVEN/AN/AN/AN/AN/A7058.443−F5.5IVE21431.911.3122.58240.644−F0.2IIINone6375.442.936.82351.345−F0.2IIIE11463.21.914.48197.146−F1.4IIIE7452.91.715.57399.351−F0.0IINone4356.33.15.63769.262−M0.9IIB and Neb I8693.71.818.64577.984−M1.4IIB18512.71.318.94505.4104−F0.4IIINone6412.81.614.66282.3114−F0.3IIIE6301.91.315.85125.5115−F0.1IIIIV I11552.41.322.92993.220+F6.5IIIB and E12484.092.2611.74904.360+F1.2IIE5663.22.220.66518.094+M0.0IIIE11703.31.921.24157.2112+M0.3IINone4713.91.818.21861.7140+F0.0IIIB65431.618.03312.3153+F0.2IIIB22514.62.211.14679.4162+M0.1IVIV I175421.127.06329.5269UnM1.5IIIB10764.72.316.22141.0288UnF0.0IIINone0503.32.315.22264.3291UnF0.3IIINone4653.72.417.62405.7301UnF0.8IIAmbisentan054747.71662.3302UnF5.8IIISixsenran93052.66.03852.6304UnF1.0IIIS and E16551.91.228.98987.6311UnF1.8IIIB16623.21.719.42434.7326UnM0.2IIBN/AN/AN/AN/AN/A3689.4327UnM0.8IVNeb I5603.41.917.61600.6348UnM2.7IIN/A96242.315.53703.9371UnM1.6IIIB and Triumph6583.51.916.64033.9377UnF0.0IIINone8533.422.0315.54777.4395UnF2.7IIRemodulin8492.51.619.68773.7423UnF0.7IIIS117231.924.02764.7428UnM1.2IIBN/AN/AN/AN/AN/A10470.8442UnF0.1IIIS and Neb I18805.07215.83852.6443UnF0.3IIB2704.12.817.12724.8450UnF0.0IIBN/AN/AN/AN/AN/A4342.2CI, cardiac index (L/min/m2); CO, cardiac output (L/min); N/A, not available; OPG, circulating serum levels of osteoprotegerin (pg/ml); RAP, right atrial pressure (mmHg); TPR, total pulmonary resistance (mmHg/L/min); Un, unknown.* Denotes time from first presentation to sample time in years.† World Health Organization (WHO) Functional Class for Pulmonary Arterial Hypertension.‡ Treatments for PAH were Epoprostenol (E), intravenous Iloprost (IV I), nebulized Iloprost (Neb I), Bosentan (B), and Sildenafil (S). Open table in a new tab Recombinant OPG Induces Proliferation and Migration of PA-SMCsHaving shown that OPG is present within IPAH lesions and increased in IPAH serum, we next looked to determine whether the addition of recombinant OPG had any effect on PA-SMC proliferation and migration. Proliferation as measured by fold increase in tritiated thymidine incorporation increased >2-fold (P < 0.05) with 10 ng/ml platelet-derived growth factor (PDGF)-BB as a positive control. Recombinant OPG (1–100 ng/ml) induced proliferation in a dose-dependent manner with significance reached at 50 ng/ml (P < 0.05) (Figure 3A). These effects of OPG on tritiated thymidine incorporation were mirrored by a significant 30% increase in cell number after 72 hours. Migration of serum-starved PA-SMCs was similarly increased twofold with 10 ng/ml PDGF-BB. Recombinant OPG (1–100 ng/ml) induced a dose-dependent increase in migration, maximal at 50 ng/ml (P < 0.01) (Figure 3B).Figure 3Recombinant 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reduced BMP-R2 Expression Is Associated with Increased OPG SecretionWe next investigated whether signaling pathways implicated in PAH pathogenesis regulate OPG expression/secretion in human PA-SMCs in vitro, beginning with the BMP-R2 pathway. In an effort to mimic the loss of function of BMP-R2 caused be the heterozygous mutation,2Lane K Machado R Pauciulo M Thomson J Philips III, JA Loyd JE Nichols W Trembath R Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension.Nat Genet. 2000; 26: 81-84Crossref PubMed Scopus (1208) Google Scholar we used an siRNA oligo targeting BMP-R2. BMP-R2 expression levels were reduced by <80% after transfection with siRNA oligonucleotides targeting BMP-R2 compared with control cells, whereas transfection with a control siRNA oligo, Cyclophilin B, had no significant effect on BMP-R2 expression (Figure 4A). OPG was barely detectable in the culture medium of quiescent, unstimulated, untransfected human PA-SMCs. Transfection with siRNA targeting BMP-R2 increased the concentration of secreted OPG by fourfold (P < 0.05) (Figure 4B), whereas transfection with siRNA targeting the control gene Cyclophilin B had no significant effect.Figure 4Reduced 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)5-HT Increases OPG Secretion in a SERT-Dependent MannerOPG secretion by quiescent PA-SMCs was increased by 5-HT in a dose-dependent manner with maximal stimulation seen at 1 μmol/L (P < 0.05) (Figure 5A). To determine whether SERT activity was required for the effect of 5-HT on OPG secretion, cells were pre-incubated with 5 μmol/L fluoxetine or dimethyl sulfoxide vehicle.23Eddahibi S Raffestin B Hamon M Adnot S Is the serotonin transporter involved in the pathogenesis of pulmonary hypertension?.J Lab Clin Med. 2002; 139: 194-201Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Treatment with dimethyl sulfoxide alone had no effect on 5-HT-induced OPG secretion, whereas pre-incubation with fluoxetine significantly reduced OPG secretion after treatment with 1 μmol/L 5-HT (Figure 5B).Figure 55-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" @default.
• W2040642173 created "2016-06-24" @default.
• W2040642173 creator A5000276860 @default.
• W2040642173 creator A5001330227 @default.
• W2040642173 creator A5007560830 @default.
• W2040642173 creator A5024939054 @default.
• W2040642173 creator A5031568302 @default.
• W2040642173 creator A5070799438 @default.
• W2040642173 creator A5080765366 @default.
• W2040642173 creator A5081385074 @default.
• W2040642173 creator A5088307434 @default.
• W2040642173 creator A5090552438 @default.
• W2040642173 date "2008-01-01" @default.
• W2040642173 modified "2023-10-15" @default.
• W2040642173 title "Evidence of a Role for Osteoprotegerin in the Pathogenesis of Pulmonary Arterial Hypertension" @default.
• W2040642173 cites W1606441445 @default.
• W2040642173 cites W1963619628 @default.
• W2040642173 cites W1966970126 @default.
• W2040642173 cites W1973743865 @default.
• W2040642173 cites W1974659965 @default.
• W2040642173 cites W1982532602 @default.
• W2040642173 cites W1986398321 @default.
• W2040642173 cites W1988168296 @default.
• W2040642173 cites W1994632041 @default.
• W2040642173 cites W2000647744 @default.
• W2040642173 cites W2020798879 @default.
• W2040642173 cites W2021323060 @default.
• W2040642173 cites W2026469629 @default.
• W2040642173 cites W2038204617 @default.
• W2040642173 cites W2043992170 @default.
• W2040642173 cites W2050104200 @default.
• W2040642173 cites W2052522225 @default.
• W2040642173 cites W2065134534 @default.
• W2040642173 cites W2081291016 @default.
• W2040642173 cites W2083998789 @default.
• W2040642173 cites W2088829415 @default.
• W2040642173 cites W2107861793 @default.
• W2040642173 cites W2117963070 @default.
• W2040642173 cites W2121120992 @default.
• W2040642173 cites W2127547979 @default.
• W2040642173 cites W2127703801 @default.
• W2040642173 cites W2129651548 @default.
• W2040642173 cites W2137884826 @default.
• W2040642173 cites W2141306851 @default.
• W2040642173 cites W2145656661 @default.
• W2040642173 cites W2150422194 @default.
• W2040642173 cites W2154573929 @default.
• W2040642173 cites W2159158135 @default.
• W2040642173 cites W2159234056 @default.
• W2040642173 cites W2160857176 @default.
• W2040642173 cites W2166781825 @default.
• W2040642173 cites W27064955 @default.
• W2040642173 cites W4230841938 @default.
• W2040642173 doi "https://doi.org/10.2353/ajpath.2008.070395" @default.
• W2040642173 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2189625" @default.
• W2040642173 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18156213" @default.
• W2040642173 hasPublicationYear "2008" @default.
• W2040642173 type Work @default.
• W2040642173 sameAs 2040642173 @default.
• W2040642173 citedByCount "77" @default.
• W2040642173 countsByYear W20406421732012 @default.
• W2040642173 countsByYear W20406421732013 @default.
• W2040642173 countsByYear W20406421732014 @default.
• W2040642173 countsByYear W20406421732015 @default.
• W2040642173 countsByYear W20406421732016 @default.
• W2040642173 countsByYear W20406421732017 @default.
• W2040642173 countsByYear W20406421732018 @default.
• W2040642173 countsByYear W20406421732019 @default.
• W2040642173 countsByYear W20406421732020 @default.
• W2040642173 countsByYear W20406421732021 @default.
• W2040642173 countsByYear W20406421732022 @default.
• W2040642173 countsByYear W20406421732023 @default.
• W2040642173 crossrefType "journal-article" @default.
• W2040642173 hasAuthorship W2040642173A5000276860 @default.
• W2040642173 hasAuthorship W2040642173A5001330227 @default.
• W2040642173 hasAuthorship W2040642173A5007560830 @default.
• W2040642173 hasAuthorship W2040642173A5024939054 @default.
• W2040642173 hasAuthorship W2040642173A5031568302 @default.
• W2040642173 hasAuthorship W2040642173A5070799438 @default.
• W2040642173 hasAuthorship W2040642173A5080765366 @default.
• W2040642173 hasAuthorship W2040642173A5081385074 @default.
• W2040642173 hasAuthorship W2040642173A5088307434 @default.
• W2040642173 hasAuthorship W2040642173A5090552438 @default.
• W2040642173 hasBestOaLocation W20406421732 @default.
• W2040642173 hasConcept C126322002 @default.
• W2040642173 hasConcept C142724271 @default.
• W2040642173 hasConcept C164705383 @default.
• W2040642173 hasConcept C170493617 @default.
• W2040642173 hasConcept C2780426850 @default.
• W2040642173 hasConcept C2780930700 @default.
• W2040642173 hasConcept C2780942790 @default.
• W2040642173 hasConcept C71924100 @default.
• W2040642173 hasConcept C88045685 @default.
• W2040642173 hasConceptScore W2040642173C126322002 @default.
• W2040642173 hasConceptScore W2040642173C142724271 @default.
• W2040642173 hasConceptScore W2040642173C164705383 @default.
• W2040642173 hasConceptScore W2040642173C170493617 @default.
• W2040642173 hasConceptScore W2040642173C2780426850 @default.

• next