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• W2102002094 endingPage "2029" @default.
• W2102002094 startingPage "2018" @default.
• W2102002094 abstract "Pulmonary hypertension (PH) is a life-threatening disorder that is characterized by pulmonary arterial smooth muscle cell (PASMC) hyperplasia. Until now, little was been known about early changes that underlie the manifestation of PH. To characterize these early changes, we performed whole-genome microarray analysis of lungs from mice exposed to either 24 hours hypoxia or normoxia. TrkB, a member of the tyrosine kinase receptor family, and its ligand, brain-derived neurotrophic factor (BDNF), were strongly up-regulated in hypoxic mouse lungs, as well as in arteries of patients suffering from idiopathic pulmonary arterial hypertension (IPAH). BDNF stimulation of PASMC in vitro resulted in increased proliferation, TrkB and ERK1/2 phosphorylation, and nuclear translocation of the transcription factor early growth response factor 1 (Egr-1). In addition, increased Egr-1 expression was observed in idiopathic PAH lungs. The pro-proliferative effect of BDNF was attenuated by TrkB kinase inhibitor (K252a) or ERK1/2 inhibitor (U0126) pretreatment, and by knocking down Egr-1. Consequently, we have identified the BDNF-TrkB-ERK1/2 pathway as a proproliferative signaling pathway for PASMC in PH. Interference with this pathway may thus serve as an attractive reverse remodeling approach. Pulmonary hypertension (PH) is a life-threatening disorder that is characterized by pulmonary arterial smooth muscle cell (PASMC) hyperplasia. Until now, little was been known about early changes that underlie the manifestation of PH. To characterize these early changes, we performed whole-genome microarray analysis of lungs from mice exposed to either 24 hours hypoxia or normoxia. TrkB, a member of the tyrosine kinase receptor family, and its ligand, brain-derived neurotrophic factor (BDNF), were strongly up-regulated in hypoxic mouse lungs, as well as in arteries of patients suffering from idiopathic pulmonary arterial hypertension (IPAH). BDNF stimulation of PASMC in vitro resulted in increased proliferation, TrkB and ERK1/2 phosphorylation, and nuclear translocation of the transcription factor early growth response factor 1 (Egr-1). In addition, increased Egr-1 expression was observed in idiopathic PAH lungs. The pro-proliferative effect of BDNF was attenuated by TrkB kinase inhibitor (K252a) or ERK1/2 inhibitor (U0126) pretreatment, and by knocking down Egr-1. Consequently, we have identified the BDNF-TrkB-ERK1/2 pathway as a proproliferative signaling pathway for PASMC in PH. Interference with this pathway may thus serve as an attractive reverse remodeling approach. Pulmonary hypertension (PH) is a life-threatening disease characterized by a distinct and persistent elevation of pulmonary arterial pressure, which ultimately results in right ventricular failure and death.1Badesch D.B. Champion H.C. Sanchez M.A. Hoeper M.M. Loyd J.E. Manes A. McGoon M. Naeije R. Olschewski H. Oudiz R.J. Torbicki A. Diagnosis and assessment of pulmonary arterial hypertension.J Am Coll Cardiol. 2009; 54: S55-S66Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar, 2Hoeper M.M. Barbera J.A. Channick R.N. Hassoun P.M. Lang I.M. Manes A. Martinez F.J. Naeije R. Olschewski H. Pepke-Zaba J. Redfield M.M. Robbins I.M. Souza R. Torbicki A. McGoon M. Diagnosis, assessment, and treatment of non-pulmonary arterial hypertension pulmonary hypertension.J Am Coll Cardiol. 2009; 54: S85-S96Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar PH manifests itself in various diseases, including chronic obstructive lung disease and chronic thromboembolism, but also rare idiopathic and familial forms exist.3Simonneau G. Robbins I.M. Beghetti M. Channick R.N. Delcroix M. Denton C.P. Elliott C.G. Gaine S.P. Gladwin M.T. Jing Z.C. Krowka M.J. Langleben D. Nakanishi N. Souza R. Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2009; 54: S43-S54Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar PH has a multifactorial pathogenesis, with both vasoconstriction and structural remodeling of the pulmonary vessels contributing to disease progression.3Simonneau G. Robbins I.M. Beghetti M. Channick R.N. Delcroix M. Denton C.P. Elliott C.G. Gaine S.P. Gladwin M.T. Jing Z.C. Krowka M.J. Langleben D. Nakanishi N. Souza R. Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2009; 54: S43-S54Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar Structural changes underlying the remodeling processes include abnormal endothelial cell proliferation, as well as increased migration and proliferation of smooth muscle cells (SMC) and fibroblasts.4Hassoun P.M. Mouthon L. Barbera J.A. Eddahibi S. Flores S.C. Grimminger F. Jones P.L. Maitland M.L. Michelakis E.D. Morrell N.W. Newman J.H. Rabinovitch M. Schermuly R. Stenmark K.R. Voelkel N.F. Yuan J.X. Humbert M. Inflammation, growth factors, and pulmonary vascular remodeling.J Am Coll Cardiol. 2009; 54: S10-S19Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar Enhanced proliferation of pulmonary arterial SMCs (PASMC) can arise from a dysfunctional bone morphogenic protein receptor II; however, these mutations occur in only 10% to 40% of all idiopathic pulmonary arterial hypertension (IPAH) patients.5Machado R.D. Aldred M.A. James V. Harrison R.E. Patel B. Schwalbe E.C. Gruenig E. Janssen B. Koehler R. Seeger W. Eickelberg O. Olschewski H. Elliott C.G. Glissmeyer E. Carlquist J. Kim M. Torbicki A. Fijalkowska A. Szewczyk G. Parma J. Abramowicz M.J. Galie N. Morisaki H. Kyotani S. Nakanishi N. Morisaki T. Humbert M. Simonneau G. Sitbon O. Soubrier F. Coulet F. Morrell N.W. Trembath R.C. Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension.Hum Mutat. 2006; 27: 121-132Crossref PubMed Scopus (327) Google Scholar, 6Thomson J.R. Machado R.D. Pauciulo M.W. Morgan N.V. Humbert M. Elliott G.C. Ward K. Yacoub M. Mikhail G. Rogers P. Newman J. Wheeler L. Higenbottam T. Gibbs J.S. Egan J. Crozier A. Peacock A. Allcock R. Corris P. Loyd J.E. Trembath R.C. Nichols W.C. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family.J Med Genet. 2000; 37: 741-745Crossref PubMed Google Scholar Additionally, changes in growth factor production or receptor levels have been shown to influence the development of PH. These include; platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF),7Schermuly R.T. Dony E. Ghofrani H.A. Pullamsetti S. Savai R. Roth M. Sydykov A. Lai Y.J. Weissmann N. Seeger W. Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition.J Clin Invest. 2005; 115: 2811-2821Crossref PubMed Scopus (870) Google Scholar, 8Humbert M. Monti G. Fartoukh M. Magnan A. Brenot F. Rain B. Capron F. Galanaud P. Duroux P. Simonneau G. Emilie D. Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients.Eur Respir J. 1998; 11: 554-559PubMed Google Scholar, 9Benisty J.I. McLaughlin V.V. Landzberg M.J. Rich J.D. Newburger J.W. Rich S. Folkman J. Elevated basic fibroblast growth factor levels in patients with pulmonary arterial hypertension.Chest. 2004; 126: 1255-1261Crossref PubMed Scopus (69) Google Scholar, 10Merklinger S.L. Jones P.L. Martinez E.C. Rabinovitch M. Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary hypertension.Circulation. 2005; 112: 423-431Crossref PubMed Scopus (183) Google Scholar which act as strong mitogens and chemoattractants for pulmonary SMCs.11Yu Y. Sweeney M. Zhang S. Platoshyn O. Landsberg J. Rothman A. Yuan J.X. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression.Am J Physiol Cell Physiol. 2003; 284: C316-C330Crossref PubMed Scopus (289) Google Scholar Additionally, up-regulation of the tyrosine receptor kinases PDGFR α and β has been observed in lambs with chronic intrauterine pulmonary hypertension12Balasubramaniam V. Le Cras T.D. Ivy D.D. Grover T.R. Kinsella J.P. Abman S.H. Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus.Am J Physiol Lung Cell Mol Physiol. 2003; 284: L826-L833Crossref PubMed Scopus (17) Google Scholar and already first clinical evidence for successful treatment of PH by PDGFR interference was presented.13Ghofrani H.A. Seeger W. Grimminger F. Imatinib for the treatment of pulmonary arterial hypertension.N Engl J Med. 2005; 353: 1412-1413Crossref PubMed Scopus (401) Google Scholar Another well-recognized stimulus of pulmonary vasoconstriction and consequent vascular remodeling is chronic hypoxia.14Weissmann N. Sommer N. Schermuly R.T. Ghofrani H.A. Seeger W. Grimminger F. Oxygen sensors in hypoxic pulmonary vasoconstriction.Cardiovasc Res. 2006; 71: 620-629Crossref PubMed Scopus (54) Google Scholar, 15Beppu H. Ichinose F. Kawai N. Jones R.C. Yu P.B. Zapol W.M. Miyazono K. Li E. Bloch K.D. BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia.Am J Physiol Lung Cell Mol Physiol. 2004; 287: L1241-L1247Crossref PubMed Scopus (173) Google Scholar, 16Fike C.D. Zhang Y. Kaplowitz M.R. Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.J Appl Physiol. 2005; 99: 670-676Crossref PubMed Scopus (25) Google Scholar In response to low alveolar oxygen, voltage-gated potassium channels are down-regulated, resulting in depolarization of SMCs, subsequent Ca2+ influx, and increased vasoconstriction.17Smirnov S.V. Robertson T.P. Ward J.P. Aaronson P.I. Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.Am J Physiol. 1994; 266: H365-H370PubMed Google Scholar, 18Hong Z. Weir E.K. Nelson D.P. Olschewski A. Subacute hypoxia decreases voltage-activated potassium channel expression and function in pulmonary artery myocytes.Am J Respir Cell Mol Biol. 2004; 31: 337-343Crossref PubMed Scopus (57) Google Scholar Other important effectors for hypoxic remodeling are hypoxia-inducible transcription factors (HIFs), which are known to induce a number of genes under hypoxic conditions.19Semenza G. Signal transduction to hypoxia-inducible factor 1.Biochem Pharmacol. 2002; 64: 993-998Crossref PubMed Scopus (744) Google Scholar, 20Wenger R.H. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression.FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1011) Google Scholar, 21Semenza G.L. Oxygen-regulated transcription factors and their role in pulmonary disease.Respir Res. 2000; 1: 159-162Crossref PubMed Scopus (135) Google Scholar In a previous study, we have demonstrated genes regulated after prolonged hypoxia exposure in mouse model of hypoxia-induced PH.22Kwapiszewska G. Wilhelm J. Wolff S. Laumanns I. Koenig I.R. Ziegler A. Seeger W. Bohle R.M. Weissmann N. Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.Respir Res. 2005; 6: 109Crossref PubMed Scopus (88) Google Scholar However, early genetic changes in response to hypoxia, representing possible trigger mechanisms, leading to vascular remodeling remain poorly understood. Therefore, we have undertaken whole-genome microarray analysis to investigate transcriptional changes occurring in the lung of the hypoxic mouse model during onset of pulmonary hypertension after only 24 hours of hypoxic exposure. We identified the Ntrk2 gene encoding tyrosine-related kinase B (TrkB), a member of the tyrosine kinase receptor family, as strongly up-regulated in hypoxic mouse lungs. Against the background that i) in our study, TrkB levels were also elevated during sustained hypoxic exposure, ii) TrkB expression has been previously shown to be present on human and rat vascular SMCs and endothelial cells,23Donovan M.J. Miranda R.C. Kraemer R. McCaffrey T.A. Tessarollo L. Mahadeo D. Sharif S. Kaplan D.R. Tsoulfas P. Parada L. et al.Neurotrophin and neurotrophin receptors in vascular smooth muscle cells Regulation of expression in response to injury.Am J Pathol. 1995; 147: 309-324PubMed Google Scholar, 24Donovan M.J. Lin M.I. Wiegn P. Ringstedt T. Kraemer R. Hahn R. Wang S. Ibanez C.F. Rafii S. Hempstead B.L. Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization.Development. 2000; 127: 4531-4540Crossref PubMed Google Scholar and iii) other tyrosine kinases have previously been implicated in the development of PH, we have studied in more detail the role of TrkB in vascular remodeling. Eight-week-old male BALB/c mice were exposed to normobaric normoxia [fraction of inspired oxygen (FiO2) of 0.21] and to normobaric hypoxia (FiO2 of 0.10) for 1, 7, and 21 days, respectively. Isolation and preparation of mouse lungs have been described in detail previously.22Kwapiszewska G. Wilhelm J. Wolff S. Laumanns I. Koenig I.R. Ziegler A. Seeger W. Bohle R.M. Weissmann N. Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.Respir Res. 2005; 6: 109Crossref PubMed Scopus (88) Google Scholar, 25Kwapiszewska G. Wygrecka M. Marsh L.M. Schmitt S. Trosser R. Wilhelm J. Helmus K. Eul B. Zakrzewicz A. Ghofrani H.A. Schermuly R.T. Bohle R.M. Grimminger F. Seeger W. Eickelberg O. Fink L. Weissmann N. Fhl-1, a new key protein in pulmonary hypertension.Circulation. 2008; 118: 1183-1194Crossref PubMed Scopus (67) Google Scholar The Egr-1−/− animals were a kind gift of Patrick Charnay (Ecole Normale Supérieure, IBENS, Paris Cedex, France). The BDNF+/− and TrkB+/− mice were purchased from Jackson Laboratories (Bar Harbor, ME). Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) from lung homogenate of 18 BALB/c mice per group exposed for 24 hours to normobaric normoxia (FiO2 of 0.21) and to normobaric hypoxia (FiO2 of 0.10). RNA from 6 mice each was used as a pooled sample for labeling and hybridization. Labeled cDNA was generated using Superscript II to reverse transcribe 50 μg of RNA incorporating of Cy3- and Cy5-dCTP (all reagents from Invitrogen, Karlsruhe, Germany). The labeled cDNA was purified using a PCR purification kit (Qiagen). The volume of elute was reduced from 50 μL to ∼10 μL using a centrifugal vacuum concentrator. Competitive hybridizations (hypoxia/normoxia) were performed for 18 hours in UltraHyb buffer (Ambion, Austin, TX) at 42°C on 60mer oligonucleotide microarrays (MWG 30K mouse) using the GeneTAC hybridization station (PerkinElmer, Waltham, MA). The data were analyzed using the limma package in R.26R Development Core TeamR: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria2006Google Scholar, 27Smyth G.K. Speed T. Normalization of cDNA microarray data.Methods. 2003; 31: 265-273Crossref PubMed Scopus (1482) Google Scholar Intensity values were background subtracted; log ratios were normalized using a loess correction on the MA-plot values.27Smyth G.K. Speed T. Normalization of cDNA microarray data.Methods. 2003; 31: 265-273Crossref PubMed Scopus (1482) Google Scholar Regulated genes were filtered by using moderated t-statistics and adjusting the false discovery rate at 10%.27Smyth G.K. Speed T. Normalization of cDNA microarray data.Methods. 2003; 31: 265-273Crossref PubMed Scopus (1482) Google Scholar Experimental design is provided in Supplemental Figure S1 (available at http://ajp.amjpathol.org). Pathways were analyzed using PathwayExpress and OntoExpress of the Onto-Tools.28Khatri P. Bhavsar P. Bawa G. Draghici S. Onto-Tools: an ensemble of web-accessible, ontology-based tools for the functional design and interpretation of high-throughput gene expression experiments.Nucleic Acids Res. 2004; 32: W449-W456Crossref PubMed Scopus (124) Google Scholar Mice exposed to hypoxia (n = 10) or normoxia (n = 10) for 21 days were euthanized with ketamine (6 mg/100 g) and xylazine (1 mg/100 g) intraperitoneally. Major pulmonary arteries were dissected for isolation of mRNA and expression analysis. Lung homogenate samples from the monocrotaline-induced as well as the Su5416 rat model29Taraseviciene-Stewart L. Kasahara Y. Alger L. Hirth P. Mc Mahon G. Waltenberger J. Voelkel N.F. Tuder R.M. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension.FASEB J. 2001; 15: 427-438Crossref PubMed Scopus (629) Google Scholar of pulmonary hypertension were obtained as described previously.25Kwapiszewska G. Wygrecka M. Marsh L.M. Schmitt S. Trosser R. Wilhelm J. Helmus K. Eul B. Zakrzewicz A. Ghofrani H.A. Schermuly R.T. Bohle R.M. Grimminger F. Seeger W. Eickelberg O. Fink L. Weissmann N. Fhl-1, a new key protein in pulmonary hypertension.Circulation. 2008; 118: 1183-1194Crossref PubMed Scopus (67) Google Scholar, 30Dumitrascu R. Weissmann N. Ghofrani H.A. Dony E. Beuerlein K. Schmidt H. Stasch J.P. Gnoth M.J. Seeger W. Grimminger F. Schermuly R.T. Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling.Circulation. 2006; 113: 286-295Crossref PubMed Scopus (191) Google Scholar Samples from human lung tissue were obtained from five donors and five IPAH patients undergoing lung transplantation as described previously.31Kwapiszewska G. Markart P. Dahal B.K. Kojonazarov B. Marsh L.M. Schermuly R.T. Taube C. Meinhardt A. Ghofrani H.A. Steinhoff M. Seeger W. Preissner K.T. Olschewski A. Weissmann N. Wygrecka M. PAR-2 inhibition reverses experimental pulmonary hypertension.Circ Res. 2012; 110: 1179-1191Crossref PubMed Scopus (53) Google Scholar Patient characteristics are provided in previous publications.31Kwapiszewska G. Markart P. Dahal B.K. Kojonazarov B. Marsh L.M. Schermuly R.T. Taube C. Meinhardt A. Ghofrani H.A. Steinhoff M. Seeger W. Preissner K.T. Olschewski A. Weissmann N. Wygrecka M. PAR-2 inhibition reverses experimental pulmonary hypertension.Circ Res. 2012; 110: 1179-1191Crossref PubMed Scopus (53) Google Scholar, 32Schermuly R.T. Pullamsetti S.S. Kwapiszewska G. Dumitrascu R. Tian X. Weissmann N. Ghofrani H.A. Kaulen C. Dunkern T. Schudt C. Voswinckel R. Zhou J. Samidurai A. Klepetko W. Paddenberg R. Kummer W. Seeger W. Grimminger F. Phosphodiesterase 1 upregulation in pulmonary arterial hypertension: target for reverse-remodeling therapy.Circulation. 2007; 115: 2331-2339Crossref PubMed Scopus (120) Google Scholar Microdissection of pulmonary arteries (40 vessels per animal with a diameter of 100 to 250 μm, four animals each) was performed on mounted cryosections using the Laser Microbeam System (P.A.L.M., Bernried, Germany) as described in detail previously.22Kwapiszewska G. Wilhelm J. Wolff S. Laumanns I. Koenig I.R. Ziegler A. Seeger W. Bohle R.M. Weissmann N. Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.Respir Res. 2005; 6: 109Crossref PubMed Scopus (88) Google Scholar All immunohistochemical experiments were performed on paraffin-embedded serial slides. All antibodies were diluted (1:100) in ChemMate Antibody Diluent, (Dako, Glostrup, Denmark): anti-TrkB, anti-BDNF, anti-NT-4/5, anti-Egr-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA). After overnight incubation in a humid chamber, slides were washed three times in Tris-buffered saline (pH 7.4 to 7.6) and incubated for 45 minutes with corresponding secondary antibodies conjugated with alkaline phosphatase (Rockland, Gilbertsville, PA) at a 1:250 dilution. Negative controls were prepared with the omission of the primary antibody. For immunofluorescence analysis, untreated or BDNF treated PASMC were grown on glass slides and fixed for 5 minutes in cold methanol. Cells were then blocked for 2 hours with 3% bovine serum albumin in Tris-buffered saline, and incubated overnight at 4°C with anti-TrkB, anti-BDNF, or anti-Egr-1 antibody (all from Santa Cruz Biotechnology). Slides were then incubated with anti-rabbit IgG-Alexa 555 and mounted with fluorescence Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany). Cell nuclei were counterstained with DAPI (Sigma-Aldrich, Munich, Germany). For microscopic inspection, a Leica DMR microscope (Leica Microsystems, Wetzlar, Germany) was used. Cell lysates from lung homogenates and PASMC were separated by 12% SDS polyacrylamide gel, followed by electrotransfer to a 0.45-μm polyvinylidene difluoride membrane (ImmobilonTM-P; Millipore, Bedford, MA). After blocking with 5% nonfat dry milk in Tris-buffered saline + 0.1% Tween20, the membrane was incubated overnight at 4°C with one of the following primary antibodies: anti-BDNF, anti-TrkB, anti–Egr-1 (all from Santa Cruz Biotechnology), or anti–β-actin (Biozol, Eching, Germany). After incubation for 1 hour with horseradish peroxidase–labeled secondary antibodies (Pierce Biotechnology, Rockford, IL; all at 1:3000 in blocking solution) signals were visualized by the ECL detection system (Pierce Biotechnology). For phosphorylation study, a rabbit anti–phospho-TrkB(Tyr516)/A(Tyr490) (Santa Cruz Biotechnology), or a rabbit anti–phospho-p42/44 MAP kinase antibody (Cell Signaling Technology, Boston, MA) was applied. Blots were stripped and reprobed using a rabbit anti-TrkB (Santa Cruz Biotechnology), or an anti–p42/44 MAP kinase antibody, respectively (Cell Signaling Technology). RNA from laser-microdissected material was isolated by RNeasy Micro Kit (Qiagen), whereas RNA from lung tissues and cell culture experiments was isolated by RNeasy Mini Kit (Qiagen) according to the manufacturer's protocols. Quality and quantity of RNA was determined by 1.5% (m/v) agarose gel and UV absorbance (Nanodrop spectrophotometer; Thermo Scientific, Wilmington, DE). RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (Applied Biosystems, Foster City, CA). Real-time PCR was performed in an ABI 7900HT Sequence Detection System (Applied Biosystems) using SYBR-Green I (Qiagen) as fluorogenic probe. Primer pairs were as follows, mouse: BDNF: forward, 5′-TTCGAGAGGTCTGACGACGAC-3`, reverse, 5`-ACCCGGGAAGTGTACAAGTCC-3′; PBGD: forward, 5′-GGTACAAGGCTTTCAGCATCGC-3`, reverse, 5`-ATGTCCGGTAACGGCGGC-3′; TrkB: forward, 5′-GCAAACCAGAAAAGGCTAGAAATC-3`, reverse, 5`-TGTAAGCCACAAACTTTAAGCCG-3′; human: BDNF: forward, 5′-CAAGGCAGGTTCAAGAGGCTT-3`, reverse, 5`-CTGGACGTGTACAAGTCTGCG-3′; CCND1: forward, 5′-CCGTCCATGCGGAAGATC-3`, reverse, 5`-TAGTTCATGGCCAGCGGG-3′; Egr-1: forward, 5′-GTTTGCCAGGAGCGATGAAC-3`, reverse, 5`-CCGAAGAGGCCACAACACTT-3′; FN1: forward, 5′-CCGACCAGAAGTTTGGGTTCT-3`, reverse, 5`-CAATGCGGTACATGACCCCT-3′; PBGD: forward, 5′-CCCACGCGAATCACTCTCAT-3`, reverse, 5`-TGTCTGGTAACGGCAATGCG-3′; TrkB: forward, 5′-ACATTTCCGTCACCTTGACTTGT-3`, reverse, 5`-GGATGGATTTAGCCTCTTGGAG-3′. Each gene was measured in duplicate in at least three independent experiments. The ΔCT values (ΔCT = CTreference − CTtarget) for each target gene were calculated using PBGD as the reference gene. Primary human PASMC were purchased from Lonza (Basel, Switzerland) or PromoCell (Heidelberg, Germany) or isolated from human pulmonary arteries from nontransplantable donor lungs, as well as from lungs explanted from IPAH patients. The purity of PASMC cultures was confirmed using indirect immunofluorescence antibody staining for smooth muscle–specific isoforms of α-actin and myosin (minimum 95% of cells stained positive) and lack of staining for von Willebrand factor. Recombinant human BDNF (R&D Systems, Wiesbaden, Germany) was used at a final concentration of 5 or 10 ng/mL, ERK1/2 kinase inhibitor U0126 (final concentration 20 mmol/L) were obtained from Alamone Labs (Jerusalem, Israel) and Calbiochem (Darmstadt, Germany), respectively. The BDNF blocking antibody was purchased from Chemicon International (Temecula, CA; final concentration 2.5 μg/mL). The electrophoretic mobility shift assay was performed with nuclear extracts (Pierce Biotechnology) from human PASMC using the oligonucleotide probe (5′-GGATCCAGCGGGGGCGAGCGGGGGCGA-3′) containing the Egr consensus sequence (Santa Cruz Biotechnology) as described previously.25Kwapiszewska G. Wygrecka M. Marsh L.M. Schmitt S. Trosser R. Wilhelm J. Helmus K. Eul B. Zakrzewicz A. Ghofrani H.A. Schermuly R.T. Bohle R.M. Grimminger F. Seeger W. Eickelberg O. Fink L. Weissmann N. Fhl-1, a new key protein in pulmonary hypertension.Circulation. 2008; 118: 1183-1194Crossref PubMed Scopus (67) Google Scholar Cell proliferation of PASMC was assessed by [3H]-thymidine incorporation as described previously.31Kwapiszewska G. Markart P. Dahal B.K. Kojonazarov B. Marsh L.M. Schermuly R.T. Taube C. Meinhardt A. Ghofrani H.A. Steinhoff M. Seeger W. Preissner K.T. Olschewski A. Weissmann N. Wygrecka M. PAR-2 inhibition reverses experimental pulmonary hypertension.Circ Res. 2012; 110: 1179-1191Crossref PubMed Scopus (53) Google Scholar For siRNA studies, proliferation was measured by bromodeoxyuridine incorporation according to the manufacturer's protocol (Roche, Mannheim, Germany). Identification of apoptotic cells after 1 and 24 hours of BDNF (10 ng/mL) treatment was performed using allophycocyanin-conjugated annexin V (Invitrogen), following the recommendations of the manufacturer. Necrotic cells were excluded by counterstaining with 2 μg/mL propidium iodide. Data were collected using a FACSCanto flow cytometer and analyzed using a FACSDiva software package (both from BD Biosciences, Heidelberg, Germany). A minimum of 10,000 cells were analyzed per sample. Gates based on forward and side scatter were set to eliminate cellular debris and cell clusters. PASMC were transfected with small interference RNA (siRNA) at (100 nmol/L) using X-treme Gene siRNA Transfection Reagent (Roche) or Lipofectamine2000 (Invitrogen). Predesigned, commercially available siRNA against human Egr-1 (siGenomeSMART pool) was purchased from Dharmacon (Chicago, IL), TrkB (siGenomeSMART pool) from Dharmacon and Santa Cruz Biotechnology, siRNA against BDNF (Santa Cruz Biotechnology), control siRNA from Ambion. Knockdown efficiency was assessed 24 hours (Egr-1) or 72 hours (TrkB and BDNF) post transfection by real-time PCR. Bar charts show mean ± SEM. Differences between more than two groups were assessed by analysis of variance followed by Dunnett's multiple-to-one comparison post hoc tests. All data that were subjected to statistical analysis were checked for normal distribution by quantile–quantile plots. Where appropriate, the paired Student's t-test was performed. All experiments were designed with matched control conditions to enable statistical comparison. An asterisk indicates P ≤ 0.05. RNA expression profiles were compared from lung homogenates from mice exposed to normoxia or hypoxia for 24 hours using whole-genome microarray analysis. Under hypoxia, 918 genes were up-regulated and 648 genes were down-regulated (10% false discovery rate). Among these genes, 99 were up- and 27 down-regulated by fold change ≥2. Genes previously reported to be involved in hypoxic remodeling [ie, PDGFb, vascular endothelial growth factor (VEGF) c, and endothelin1 (Edn1)] revealed strong up-regulation following hypoxia exposure (Figure 1A). Several gene pathways were overrepresented in the candidate lists (Figure 1B). Of interest, genes involved in the MAPK signaling pathway revealed strong regulation, where the strongest induction was observed for Ntrk2/TrkB (∼sevenfold up). Because tyrosine kinases have previously been implicated in the development of PH, we therefore studied in more detail the role of TrkB in vascular remodeling. We subsequently investigated the expression of TrkB at more time points by real-time PCR. Short exposure to hypoxia (24 hours) strongly induced TrkB expression, confirming the array data (Figure 1C). High levels of TrkB expression were maintained during prolonged exposure of mice to hypoxia (7 and 21 days). To localize the expression of TrkB and its ligands, BDNF and neurotrophin 4 (NT-4) in the lung parenchyma, immunohistochemical staining was performed. All three proteins localized to epithelial cells in the bronchial tree; however, TrkB and BDNF were also localized to the pulmonary vasculature as indicated by smooth muscle actin (SMA) staining of consecutive sections (Figure 1D; see also Supplemental Figure S2 at http://ajp.amjpathol.org). Antibody specificity was assessed on BDNF and TrkB knockout mice tissues (see Supplemental Figure S3 at http://ajp.amjpathol.org). To determine TrkB- and BDNF-specific expression in PH, we isolated major pulmonary arteries from mice exposed to either 21 days hypoxia or normoxia. Similar to the induction observed in whole lungs, increased expression of TrkB was observed in pulmonary arteries (Figure 1E). Interestingly, increased BDNF expression was also detected (Figure 1E). Laser-microdissection of intrapulmonary arteries in combination with real-time PCR showed similar induction of TrkB and BDNF expression under hypoxic conditions (Figure 1F). Expression of TrkB and BDNF by SMC was confirmed by real-time PCR (Figure 1G), and immunofluorescence staining of isolated mouse PASMC (Figure 1H) and microvascular pulmonary SMCs (see Supplemental Figure S4A at http://ajp.amjpatho" @default.
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