BDNF/TrkB Signaling Augments Smooth Muscle Cell Proliferation in Pulmonary Hypertension  Grazyna Kwapiszewska, Karolina Chwalek, Leigh M. Marsh, Malgorzata Wygrecka, Jochen Wilhelm, Johannes Best, Bakytbek Egemnazarov, Friederike C. Weisel, Sarah L. Osswald, Ralph T. Schermuly, Andrea Olschewski, Werner Seeger, Norbert Weissmann, Oliver Eickelberg, Ludger Fink  The American Journal of Pathology  Volume 181, Issue 6, Pages 2018-2029 (December 2012) DOI: 10.1016/j.ajpath.2012.08.028 Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 1 Ntrk2 is up-regulated in mouse lungs after 24 hours of hypoxia exposure (pO2 = 0.01). Total RNA from mice lung homogenates was isolated (n = 18 per group), direct labeled, and hybridized to 44K 60mer oligonucleotide microarrays (MWG). A: Volcano plot: log2-fold regulation compared to probability of regulation. Genes with log odds values ≥5 were considered to be regulated; black spots, exemplary genes. B: Functional annotation of regulated genes was performed according to Gene Ontology (GO). Ntrk2 (TrkB) belongs to MAPK signaling pathway. C: A time course of TrkB expression was performed using real-time PCR of lung homogenates harvested 1, 7, or 21 days after hypoxia exposure (n = 7 to 9, each). D: Immunohistochemical staining of TrkB and its ligand, BDNF in mouse lung sections. The arrow indicates positive staining. E and F: Real-time PCR analysis of TrkB and BDNF expression in (E) isolated major pulmonary arteries (n = 6), and (F) laser-microdissected intrapulmonary vessels (40 vessels per animal, n = 4), from mice exposed to 21 days of hypoxia or normoxia. G: Expression levels of TrkB and BDNF in mouse PASMC as indicated by real-time PCR; lower CT values represent a more abundant transcript level. H: Immunofluorescence staining of TrkB and BDNF in mouse PASMC. The arrows indicate TrkB localization. Inset, negative control staining. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 2 Analysis of TrkB and BDNF expression in rat PAH models. A: Real-time PCR analysis of TrkB and BDNF mRNA expression 14 and 28 days after monocrotaline administration (n = 4 per group) in lung homogenates. B: Immunohistochemical staining of TrkB and BDNF in monocrotaline-treated lungs and lungs from control animals. C: Real-time PCR analysis of TrkB and BDNF mRNA expression in lung homogenates in the Su5416 rat model (n = 4 controls, n = 7 Su5416). n.s., not significant. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 3 Up-regulation of TrkB and BDNF in human lung tissue from IPAH. A: Colocalization of BDNF and TrkB with SMA as indicated by immunofluorescence staining in donor and IPAH patients. B: Real-time PCR analysis of TrkB and BDNF in laser-microdissected intrapulmonary vessels from donor and IPAH patients. Expression is given in comparison to samples from donor lungs (n = 5 per group). C: Real-time PCR analysis of expression levels of TrkB and BDNF in human PASMC; lower CT values represent a more abundant transcript level. D: Immunofluorescence staining of TrkB and BDNF in human PASMC derived from donor and IPAH patients. E: Colocalization of BDNF and TrkB with SMA in PASMC as indicated by immunofluorescence staining, IgG, isotype control staining. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 4 Functional role of TrkB and its ligand BDNF in remodeling. Proliferation after BDNF stimulation was assessed by direct [3H] thymidine incorporation (A) (n = 6), or immunofluorescence staining of Ki-67 proliferation marker (B). The effect of BDNF on cell apoptosis was measured by annexin V staining (C) and caspase-3 cleavage (D). 5% FCS and staurosporine were used as positive controls for proliferation and apoptosis studies, respectively. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 5 BDNF stimulation leads to MAP kinase activation. A: Time-dependent phosphorylation in response to BDNF of the ERK1/2 kinase with densitometric analysis. B and C: Effects on BDNF signaling by pretreatment with the inhibitors (B) K252a (TrkB) and (C) U0126 (ERK1/2) with densitometric analysis. Equal protein loading was confirmed by immunoblotting against β-actin. D: Effects of blocking BDNF antibody on ERK1/2 phosphorylation with densitometric analysis. M(kDa), molecular weight in kilodaltons; n.s., not significant. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 6 BDNF regulates and activates Egr-1. A: Induction of early growth-response factor 1 (Egr-1) protein in human PASMC as analyzed by Western blotting. B: BDNF-induced Egr-1 protein localization in PASMC as assessed by immunofluorescence analysis. C: Electrophoretic mobility shift assay for Egr-1 induction and DNA–protein interactions from nuclear extracts prepared from BDNF (10 ng/mL)-treated PASMC, incubated with radiolabeled oligonucleotide containing Egr-1 consensus sequence. Incubation with an excess (10-fold) of an unlabeled probe served as a control for specific binding. PDGF (10 ng/mL) treated cells served as a positive control. D: Egr-1 expression in lung parenchyma of donor and IPAH patients. An Egr-1−/− mouse (KO-Egr-1) served as negative control. E: Real-time PCR analysis for Egr-1 expression in IPAH patient and donor laser-microdissected intrapulmonary vessels (n = 5). n.s., not significant. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions

Figure 7 BDNF-induced proliferation in human PASMC associates with an activation of MAP kinases and Egr-1. A: Consequence of BDNF (10 ng/mL) treatment on expression of Egr-1, cyclin D1, and fibronectin in the presence of K252a (TrkB inhibitor) and U0126 (ERK inhibitor), as assessed by real-time PCR (n = 3). Effects of (B) K252a and U0126 and (C) Egr-1 knockdown, on BDNF-induced proliferation in human primary PASMC as determined by direct [3H] thymidine or bromodeoxyuridine incorporation (n = 3). n.s., not significant. *P ≤ 0.05. The American Journal of Pathology 2012 181, 2018-2029DOI: (10.1016/j.ajpath.2012.08.028) Copyright © 2012 American Society for Investigative Pathology Terms and Conditions