Imatinib Attenuates Hypoxia-induced Pulmonary ... - ATS Journals

Imatinib Attenuates Hypoxia-induced Pulmonary Arterial Hypertension Pathology via Reduction in 5-Hydroxytryptamine through Inhibition of Tryptophan Hydroxylase 1 Expression Loredana Ciuclan1, Martin J. Hussey1, Victoria Burton1, Robert Good1, Nicholas Duggan1, Sarah Beach1, Peter Jones1, Roy Fox1, Ieuan Clay2, Olivier Bonneau1, Irena Konstantinova1, Andrew Pearce1, David J. Rowlands1, Gabor Jarai1, John Westwick1, Margaret R. MacLean3, and Matthew Thomas1 1

Respiratory Disease Area, Novartis Institutes for BioMedical Research, Horsham, West Sussex, United Kingdom; 2Epigenetics, Novartis Institutes for BioMedical Research, Basel, Switzerland; and 3College of Medical, Veterinary, and Life Sciences, Research Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom

Rationale: Whether idiopathic, familial, or secondary to another disease, pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, neointimal hyperplasia, medial hypertrophy, and adventitial fibrosis. Imatinib, a potent receptor tyrosine kinase inhibitor, reverses pulmonary remodeling in animal models of PAH and improves hemodynamics and exercise capacity in selected patients with PAH. Objectives: Here we use both imatinib and knockout animals to determine the relationship between platelet-derived growth factor receptor (PDGFR) and serotonin signaling and investigate the PAH pathologies each mediates. Methods: We investigated the effects of imatinib (100 mg/kg) on hemodynamics, vascular remodeling, and downstream molecular signatures in the chronic hypoxia/SU5416 murine model of PAH. Measurements and Main Results: Treatment with imatinib reduced all measures of PAH pathology observed in hypoxia/SU5416 mice. In addition, 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) expression were reduced compared with the normoxia/ SU5416 control group. Imatinib attenuated hypoxia-induced increases in Tph1 expression in pulmonary endothelial cells in vitro via inhibition of the PDGFR-b pathway. To better understand the consequences of this novel mode of action for imatinib, we examined the development of PAH after hypoxic/SU5416 exposure in Tph1deficient mice (Tph12/2). The extensive changes in pulmonary vascular remodeling and hemodynamics in response to hypoxia/SU5416 were attenuated in Tph12/2 mice and further decreased after imatinib treatment. However, imatinib did not significantly further impact collagen deposition and collagen 3a1 expression in hypoxic Tph12/2 mice. Post hoc subgroup analysis suggests that patients with PAH with greater

(Received in original form June 8, 2012; accepted in final form September 26, 2012) L.C. is the recipient of a Novartis Institutes for BioMedical Research Postdoctoral Fellowship. Author Contributions: L.C. designed and performed the experiments, analyzed the data, and wrote the manuscript. M.J.H., N.D., V.B., R.G., S.B., P.J., R.F., I.K., O.B., and D.J.R. provided help performing the in vivo and in vitro experiments. A.P., M.R.M., G.J., and J.W. provided intellectual input. M.T. provided project supervision. Correspondence and requests for reprints should be addressed to Matthew Thomas, Ph.D., Respiratory Disease Area, Novartis Institutes for BioMedical Research, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK. E-mail: [email protected] The article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 187, Iss. 1, pp 78–89, Jan 1, 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201206-1028OC on October 18, 2012 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Imatinib mesylate (QTI571) is an emerging therapy for pulmonary arterial hypertension (PAH) whose principal mechanism of action is via inhibition of tyrosine kinase (TK) associated with the receptor for platelet-derived growth factor (PDGF). PDGF receptor (PDGFR) signaling, vascular lesion association, and disease efficacy through inhibition with imatinib have been demonstrated preclinically in multiple models of PAH. However, attempts to reconcile the profound preclinical and clinical efficacy observed after imatinib treatment has led to further investigation of other potential mechanisms of action. What This Study Adds to the Field

Here we investigate interplay between PDGFR signaling and tryptophan hydroxylase 1 (TPH1) expression, discovering a novel mechanism of action for imatinib within a hypoxia/SU5416 preclinical model of experimental PAH. The established murine model displays many of the hallmarks of the human disease, providing a system to study the treatment and molecular mechanisms responsible for PAH. Downstream cellular and molecular analyses provide a mechanism for TPH1-dependent imatinib inhibition of perivascular fibrosis.

hemodynamic impairment showed significantly reduced 5-HT plasma levels after imatinib treatment compared with placebo. Conclusions: We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-b signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagenmediated mechanisms of fibrosis. Keywords: pulmonary arterial hypertension; animal preclinical models; imatinib; 5-hydroxytryptamine; platelet-derived growth factor receptor

The pathogenesis of pulmonary arterial hypertension (PAH) is a complex multicellular disease of increased vascular tone and remodeling, in which fibroblasts, smooth muscle, endothelial cells, and platelets play a role (1). The resultant increases in pulmonary vascular resistance and impedance cause pressure and volume overloading of the right ventricle (RV), leading to RV failure and death (2). Current therapies address vasoconstrictive elements of the disease yet provide only modest benefits on disease

Ciuclan, Hussey, Burton, et al.: Interplay between PDGF and 5-HT Pathways within PAH

progression (3). Further understanding of the vascular remodeling process may provide opportunities for more effective therapies (4). The pro-proliferative, apoptosis-resistant phenotype of smooth muscle cells (SMCs), endothelium, and fibroblasts observed in vascular lesions have been accredited to a perturbation of a number of mechanisms, including inflammatory mediators, progenitor cell influx, metabolic signaling, vasoactive factors, and growth factors (4, 5). Imatinib mesylate (QTI571) is an emerging therapy for PAH whose principal mechanism of action is via inhibition of tyrosine kinase (TK) associated with the receptor for platelet-derived growth factor (PDGF) (6). The rationale for PDGF involvement in the pathogenesis of PAH is strong, with increased expression of ligand and phosphorylated receptor in patient lung tissue, colocalized with vascular lesions (7, 8). Produced by and acting on numerous cell types, PDGF downstream signaling results in survival, proliferation, migration, and extracellular matrix deposition (7, 9, 10). PDGF exerts biological responses via activation of two highly specific, transmembrane receptor tyrosine kinases (RTKs), termed PDGF receptor (PDGFR) a and b (4, 11). PDGFR signaling, vascular lesion association, and disease efficacy through inhibition with imatinib have been demonstrated preclinically in multiple models of PAH (11, 12). However, attempts to reconcile the profound preclinical and clinical efficacy observed after imatinib treatment has led to further investigation of other potential mechanisms of action. Imatinib is also a potent inhibitor of TKs associated with receptors for stem cell factor (c-Kit), Abelson kinase (ABL), discoidin domain receptors (DDR), and colony-stimulating factor 1 (CSF1) (13). Of these activities, c-Kit inhibition may contribute to imatinib efficacy by blocking the infiltration of pathologic Kit1 endothelial progenitor populations (7). Although no definitive roles for ABL, discoidin domain receptors, or CSF1 have been proposed in PAH, it is becoming increasingly evident that the pathways targeted by imatinib mirror the complexity of the disease (14, 15). 5-Hydroxytryptamine (5-HT) has been associated with many of the pathologic processes, cell types, and signaling cascades characteristic of PAH (16). Increased in circulation of patients with PAH, 5-HT is known to induce pulmonary vessel constriction via 5-HT receptor 5HTR1B (17, 18), to induce pulmonary artery smooth muscle cell (PASMC) proliferation via the transporter (5HTT) (19), and to negatively impact cardiac health via 5HTR2B signaling (20). Preclinical and clinical studies have demonstrated up-regulation of 5-HT network in PAH, including enhanced tryptophan hydroxylase 1 (TPH1—the rate-limiting enzyme in peripheral 5-HT synthesis) expression in pulmonary artery endothelial cells (PAECs) (21–23). Hypoxia-induced PAH is partially attenuated in Tph1–/– mice, protecting against vascular remodeling and RV pressure (RVP), yet has little or no effect on RV hypertrophy (RVH) (24). Others have demonstrated inhibition of hypoxia- and monocrotaline-induced PAH using approaches that nonspecifically inhibit both Tph1 and Tph2, such as p-chlorophenylalanine (16, 24). Recent data demonstrate transactivation of PDGFR-b by 5-HT in PASMCs leading to proliferation and migration, indicating a link between PDGF and 5-HT pathways in a PAHrelevant mechanism (25, 26). Here we investigate interplay between PDGFR signaling and TPH1 expression, discovering a novel mechanism of action for imatinib, within a hypoxia/SU5416 preclinical model of experimental PAH. The established murine model displays many of the hallmarks of the human disease, providing a robust system to study the treatment, prevention, and molecular mechanisms responsible for PAH (27). Downstream cellular and molecular analyses provide a mechanism for TPH1-dependent imatinib inhibition of perivascular fibrosis. Elements of the data have been previously reported in the form of an abstract (28).

79

METHODS Reagents See the online supplement for additional materials and methods.

Animal Models and Experimental Design All animal procedures were conducted in accordance with protocols approved by Animals Scientific Procedures Act, 1986, UK. TPH1 knockout (Tph12/2) (24) or wild-type (WT) littermate control mice were administered SU5416 (20 mg/kg) once weekly, under normoxia or hypoxia as described previously (27). Additional groups received imatinib (100 mg/kg) or placebo twice daily. At study termination, mice were killed and PAH pathology was assessed.

Assessment of PAH Hemodynamic and right ventricular hypertrophy. After treatments, RVP, RVH, and systemic blood pressure (SBP) measurements were undertaken as described (27).

Echocardiographic Assessment Cardiac parameters were assessed as previously described (27).

Histology and Immunohistochemistry Lung tissue preparation, sectioning, staining, and vascular morphometry were done as described (27). Sections were probed with anti a-smooth muscle actin (a-SMA), anti-mouse Willebrand factor, proliferating cell nuclear antigen (PCNA) antibodies, and picrosirius red (PSR) as previously described (29) (see Table E1 in the online supplement). Medial wall thickening was assessed as described elsewhere (11).

Cell Culture and Treatments 5-HT measurement assay. BON (human pancreatic carcinoid) cell line was a gift from Dr. Courtney M. Townsend (University of Texas, Galveston, TX) and cultured as previously described (30). Cells were incubated with imatinib (0.1–10 mM), and 5-HT levels were measured using AlphaLISA kit (Perkin Elmer, Cambridge, UK). Human pulmonary arterial endothelial cells. Human pulmonary arterial endothelial cells (HPAECs) were obtained from Promocell (Heidelberg, Germany) and maintained as previously described (31). Cells were preincubated with vehicle (dimethyl sulfoxide) or imatinib (10 mM) for 1 hour, then stimulated with PDGF-BB (30 ng/ml) under normoxic/ hypoxic conditions (0.1% O2) for 15 minutes to 24 hours. Small interfering RNA-mediated gene silencing. HPAECs were transfected using a scrambled nontargeting control small interfering siRNA (NTCsi) or siRNA targeting human Pdgfr-a, Pdgfr-b, and c-Kit (ONTARGETplus SMARTpool; Dharmacon, Thermo Fisher Scientific, Lafayette, CO). After the incubation period, cells were exposed to normoxic/hypoxic conditions for 15 minutes to 24 hours (31).

Protein Isolation and Immunoblotting Protein extraction from mouse lungs, BON cells, and cultured HPAECs and immunoprecipitation/Western blot analysis were performed as described previously (27, 31). Blots were probed with rabbit anti-TPH1, PDGFR-b, anti-PathScan PDGFR activity assay, and antimouse b-actin served as a loading control (Table E1).

Gene Expression Studies The extraction of RNA from cultured cells and lung homogenates, generation of cDNA, and gene expression analysis by quantitative real-time reverse transcriptase–polymerase chain reaction (qPCR) were performed as described previously (27, 31). The following gene expression assays were used: Tph1, Pdgfr, 5Htt, 5HT1b, 5HT2a, 5HT2b, a-sma, collagen 3a1 (Col3a1), Ctgf, Fn, and Gapdh (Table E2).

80

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

VOL 187

2013

Figure 1. Treatment with imatinib reduced all measures of pulmonary arterial hypertension (PAH) pathology observed in hypoxia/SU5416 mice. Imatinib (Im) effects on PAH development were determined in male C57/Bl6 mice injected subcutaneously once weekly with SU5416 (SU) (20 mg/kg) or vehicle followed by exposure to chronic normobaric hypoxia (Hx) (10% O2) in a ventilated chamber or normoxia (Nx) for 3 weeks. Additional groups of animals received twice daily either imatinib (100 mg/kg, dissolved in distilled water) or vehicle orally by gavage. Development of (A) right ventricular (RV) systolic pressure (RVP; mm Hg), (B) RV hypertrophy (RVH, Fulton index, RV/ [LV1S] weight ratio), (C) Systemic blood pressure (SBP), and (D) cardiac output (CO) (ml/min) during 21 days of Hx or Nx exposure. Data are mean 6 SEM for 10 animals per group. Statistical differences (*P , 0.05, **P , 0.01, ***P , 0.001) are indicated by analysis of variance. LV ¼ left ventricle; S ¼ septum.

In Silico Promoter Analysis Tph1 promoter sequences from mouse were retrieved and analyzed using MatInspector (http://www.genomatix.com [32, 33]). Tph1 promoter binding sites were design using NTI vector software.

human TPH1 enzyme (25 nM) and substrate solution (tryptophan [40 mM] and cofactor tetrahydrobiopterin [80 mM]). TPH1 activity was stopped with the addition of 30% H2SO4 (vol/vol) and measured on Envision (Perkin Elmer, Cambridge, UK).

In Vitro TPH1 Activity Assay

Plasma Samples and Cytokine Levels

Test compounds (imatinib and LP533401) were used to construct a 10point dose–response curve (10 mM to 0.5 nM) before incubation with

Cytokine levels in mouse platelet-rich plasma and total plasma samples were assessed using a 5-HT ELISA (Fast Track 5-HT; Labor Figure 2. Anti-remodeling potency of imatinib (Im) in pulmonary vasculature of mice exposed to chronic hypoxia under vascular endothelial growth factor receptor inhibition. (Aa–Ad). The degree of muscularization is demonstrated by von Willebrand (vWF) (brown) and a-smooth muscle actin (a-SMA) (red) staining for identifying endothelium and vascular smooth muscle cells in small pulmonary arteries after 21 days’ treatment with imatinib under normoxia (Nx)/hypoxia (Hx) environment. Representative pictures of the altered vessel phenotype in lung tissue. (Ae–Ah) For each artery, the medial wall thickness was expressed as follows: % wall thickness ¼ ([medial thickness 3 2]/external diameter) 3 100. (Ai–Al) Representative bright-field micrographs of proliferating cell nuclear antigen (PCNA)-stained lung tissue; arrows indicate positive proliferative cell staining. (Am–Ap) Representative brightfield micrographs of collagen fibers (picrosirius red [PSR]); arrows indicate perivascular distribution. Morphometric analysis for specific staining quantification of (B) vascular muscularization, (C) % medial wall thickness, (D) PCNA, and (E) PSR. (F) Effect of imatinib on occlusive pulmonary lesions in Hx/SU5416 pulmonary arterial hypertension (PAH). (G) Representative image of an a-SMA–stained occlusive pulmonary lesion in the HX/SU5416 PAH. (Nx/SU and Hx/SU mice treated with imatinib under normoxic or hypoxic exposure; 203). Results are expressed as mean 6 SEM (n ¼ 10). Statistical differences (*P , 0.05, ***P , 0.001) are indicated by analysis of variance as a star above each bar. Veh ¼ vehicle.


81

Figure 2. (Continued).

Diagnostika, Nordhorn, Germany) and an Angiogenesis Antibody Array (R&D Systems, Inc., Abingdon, UK), following the manufacturer’s instructions (27).

Study Patient Subjects Plasma samples from 25 patients within the imatinib proof of concept trial (34, 35) were assessed for 5-HT levels at baseline and after 6 months of treatment. The study included 59 patients in functional class (FC) II–IV with idiopathic or heritable PAH, or PAH associated with connective tissue disease or congenital heart disease (35).

Statistical Analysis Values are presented as mean 6 SEM for 5 to 10 animals per group. Groups were compared with a two-tailed t-test or one-way analysis of variance with Dunnett modification. Significance between groups was determined using two-way analysis of variance with Bonferonni post hoc analysis. A level of P less than 0.05 was considered significant.

RESULTS Treatment with Imatinib Reduced All Measures of PAH Pathology Observed in Hypoxia/SU5416 Mice

Moderate to severe PAH was induced via weekly injection of SU5416 (SU) combined with 3 weeks of chronic hypoxia (Hx) (27). Subgroups of mice were treated twice daily with orally administered vehicle (Veh) or 100 mg/kg imatinib (Im) (Figure 1A). Nx/SU5416-treated mice were considered as the control group for all PAH pathology measurements. In chronically hypoxic mice, PAH developed within 21 days, characterized by a significant increase in RVP as compared with normoxic (Nx/ SU5416) animals (.45 mm Hg). Treatment with imatinib reduced RVP in Hx/SU mice to near-normal levels (25–30 mm Hg, P , 0.01) (Figure 1B). In the Hx/SU groups, significant RVH developed as a consequence of increased RVP. Fulton index increased from 0.20 (Nx/ SU) to 0.31 (Hx/SU). Imatinib caused a reduction of this ratio to 0.22 (P , 0.05) (Figure 1B). SBP did not change in any of the

treatment groups (Figure 1C). In the presence of vascular endothelial growth factor receptor (VEGFR) inhibitor, Hx decreased the cardiac output (8 ml/min) compared with control conditions (18 ml/min). Imatinib-treated animals showed significant recovery in cardiac output as compared with vehicle treatment (15 ml/min, P , 0.05) (Figure 1D). In the Hx/SU-exposed animals, a dramatic increase in fully muscularized (FM) pulmonary arterioles occurred with a concomitant decrease in nonmuscularized vessels (Figures 2Aa– 2Ac and 2B). Treatment with imatinib resulted in a significant reduction in FM vessels (Figures 2Ad and 2B). Medial wall thickness of pulmonary arteries 30 to 90 mm in diameter was markedly increased in the Hx/SU5416 groups as compared with control animals. Imatinib treatment significantly reversed the increase in medial wall thickness (Figures 2Ae–2Ah and 2C). In Hx/SU-challenged mice, the number of vascular PCNAproliferating endothelial cells and SMCs were increased compared with control mice (Figures 2Ai–2Al and 2D; see Figure E1F) (27). In parallel to normalization of vessel morphology, the number of PCNA-positive cells was considerably reduced in animals treated with imatinib (Figures 2Ak–2Al and 2D). PSR staining showed striking collagen deposition in the vessel adventitia from Hx/SU animals compared with normoxic control mice (Figures 2Am–2Ap and 2E). Quantitative histology analysis demonstrated a significantly reduced fibrotic deposition in imatinib-treated Hx mice (Figures 2Al and 2D). Imatinib had a significant effect on occlusive lesion formation, with the number of fully occluded vessels being significantly reduced in imatinib-dosed mice (Figures 2F and 2G). Biomarker Analysis in Response to Imatinib Treatment Revealed a Fall in Platelet 5-HT, in Accordance with Reduced Lung Tph1 Expression

Plasma analysis confirmed an increase in vasoactive molecules and growth factors as vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), monocyte chemotactic protein-1 (MCP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen

82


VOL 187

2013

Figure 3. Circulating biomarker analysis in response to imatinib reveals a fall in platelet-rich 5-hydroxytryptamine (5-HT), which correlates with a reduction in lung tryptophan hydroxylase 1 (Tph1) expression. (A) Peripheral venous blood samples were collected on ethylenediaminetetraacetic acid and centrifuged to obtain platelet-rich plasma (PRP, 200 g; 5 min). Shown are mean values of platelet-rich plasma 5-HT in vehicle (SU) and imatinib (SU/Im) groups exposed to normoxia (Nx) and hypoxia (Hx, hatched bars). ELISA data are mean 6 SEM for five animals per group, expressed in ng/ml. (B– D) Lung changes in mRNA expression of (B) Tph1, (C) 5-HT transporter (5HTT), and (D) 5HT1b, normalized to Gapdh in response to Nx or Hx plus vehicle (SU/Veh) or imatinib (SU/Im). Quantitative changes in gene expression were analyzed by quantitative real-time polymerase chain reaction (DDCt method). Each bar shows mean 6 SEM of four animals per group. (E) Representative Western blots for expression of TPH1 from lungs treated with vehicle (SU/veh) or imatinib (SU/Im) and exposed to Nx or Hx for 3 weeks. (F) Densitometry was performed using Image J Analysis software and normalized for b-actin. (G) Representative immunoprecipitation/Western blot for expression of P–platelet-derived growth factor receptor (PDGFR)-b and PDGFR from lungs treated with vehicle (SU/veh) or imatinib (SU/Im) and exposed to Nx or Hx for 3 weeks. (H) Densitometry was performed using Image J Analysis software and normalized for PDGFR. Statistical significance (*P , 0.05, **P , 0.01, ***P , 0.001) was determined using t test.

activator inhibitor 1 (PAI-1), PDGFAA/BB, granulocyte macrophage colony–stimulating factor (GM-CSF), and stromal cell– derived factor-1 (SDF-1) after hypoxia and VEGFR inhibition. Plasma VEGF, TIMP-1, MCP-1, and GM-CSF levels were reduced with imatinib administration (P , 0.05), leaving the rest unaffected (Table E1). Surprisingly, increased levels of 5-HT observed in Hx/SU-exposed mice (1,500 ng/ml), were significantly reduced in mice receiving imatinib for 21 days (600 ng/ml) (Figure 3A). To validate the pharmacological effect of imatinib on 5-HT levels, we examined components of the 5-HT signaling

system in lung tissue. In accordance with elevated 5-HT after Hx/SU exposure, increased expression of Tph1, 5-HT receptors (5HT1b, 5HT2a, 5HT2b), and 5-HT transporter (5HTT) was observed by qPCR (Figures 3B–3D; Figures E2A and E2B). Treatment with imatinib ablated the increase in Tph1 mRNA after Hx exposure (Figure 3B), leaving 5HT1b, 5HT2b, 5HT2b, and 5HTT expression unaffected (Figures 3F and 3G; Figures E2A–E2C). The pattern of Tph1 expression was reflected in Western blot analysis of whole lung lysates (Figures 3E and 3F). Imatinib attenuated Hx-induced increases in TPH1 protein expression and PDGFR-b–p44/42–mitogen-activated protein


83

Figure 4. Imatinib-attenuated hypoxiainduced increases in tryptophan hydroxylase 1 (TPH1) expression in human pulmonary arterial endothelial cells (HPAECs) in vitro via inhibition of the platelet-derived growth factor receptor (PDGFR)-b pathway. (A) Western blot analysis of cultured HPAECs pretreated with imatinib (Im; 10 mM) and then stimulated with PDGF and/or exposure to hypoxia (Hx) (1% O2), as indicated. (B) Densitometry analysis was used to determine TPH1 expression levels, normalized for b-actin. (C) Tph1 mRNA expression in cultured cells exposed to hypoxia and/or PDGF for 24 hours. Quantitative changes in gene expression were analyzed by quantitative real-time polymerase chain reaction (qPCR) (DDCt method). (D) Western blot analysis of TPH1 in HPAECs transfected with 10 nmol small interfering oligonucleotides, against control vector (siRNA NTC) or siRNA PDGFR-a,-b, and stem cell factor receptor (c-Kit) for 48 hours. Specific quantitative changes in gene expression were analyzed by qPCR (DDCt method) (Figures E6 and E7). (E) qPCR analysis was used to determine Tph1 mRNA levels in HPAECs exposed to Hx for 24 hours. (F) Densitometry to quantify TPH1 expression was performed using Image J Analysis software and normalized for b-actin. (G) qPCR analysis was used to determine PDGFR-b mRNA levels in transfected HPAECs. Data are the mean 6 SEM for two independent experiments. *P , 0.05, **P , 0.01, ***P , 0.001, compared with NTCsi-transfected HPAECs by Student t test. DMSO ¼ dimethyl sulfoxide.

kinase pathway phosphorylation (P , 0.05) (Figures 3E–3H; Figures E3A and E3B). We documented no significant differences in 5-HT levels and TPH1 expression between Nx/CTL- and Nx/SU5416-treated mice (27). It is noteworthy that the compound did not reduce brain Tph1 and Tph2 mRNA levels under hypoxic exposure (Figures E4A and E4B). Imatinib Attenuated Hypoxia-induced Increases in Tph1 Expression in HPAECs In Vitro via Inhibition of the PDGFR-b Pathway

We next investigated whether imatinib was involved in Hx-induced expression of Tph1 in HPAECs and if activation of PDGF-BB had a similar impact. Stimulation of HPAECs with PDGF-BB and Hx exposure markedly increased the activation of p44/42 mitogen-activated protein kinase (15 min) and TPH1 expression (24 h) (Figure 4A; Figures E5A and E5B).

In a similar manner with in vivo data, 1-hour pretreatment of HPAECs with imatinib abrogated PDGF-BB and/or Hx-induced activation of downstream targets (P , 0.05) (Figures 4A–4C). PDGF treatment alone resulted in a slight increase in Tph1 expression compared with dimethyl sulfoxide control mice (Figures 4B and 4C). To determine which inhibitory targets of imatinib reduced TPH1 expression, we first examined the expression of Tph1, Pdgfr-a, Pdgfr-b, c-Kit, and abl genes after Hx exposure of HPAECs. qPCR data indicate up-regulation of Tph1, Pdgfr-a, Pdgfr-b, and c-Kit mRNA from 3 hours onward, with no significant differences in abl expression (Figures E6A–E6E). No significant differences in Tph1 expression were observed after genetic silencing of Pdgfr-a and c-Kit, whereas Pdgfr-b knockdown resulted in a significant decrease in Tph1 mRNA expression and protein levels as compared with NTCsi-transfected

84


VOL 187

2013

Figure 5. Imatinib treatment decreases 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) levels in BON cells, without impacting TPH1 enzymatic activity. (A) 5-HT quantification (AlphaLISA kit) of BON cells incubated with increasing concentrations of imatinib (0.1–10 mM) at 378 C, 5% CO2 for 48 hours. (B) Lung changes in mRNA expression of Tph1 normalized to Gapdh in BON cells treated with 1.1 mM imatinib for 48 hours. All presented data are representative for two different preparations, investigated in triplicate each. Quantitative changes in gene expression were analyzed by quantitative real-time polymerase chain reaction (DDCt method). (C) Representative Western blots for expression P–platelet-derived growth factor receptor (PDGFR)-b from BON cells pretreated with vehicle (dimethyl sulfoxide) or imatinib (Im) and exposed to PDGF (30 ng/ml) for 15 minutes. Densitometry was performed using Image J Analysis software and normalized to b-actin. Statistical significance (***P , 0.001) was determined using t test. (D) TPH1 activity was measured in the presence of either imatinib (0.1–10 mM) or LP533401 (Lexicon compound) after the reaction was incubated for 25 minutes.

hypoxic control HPAECs (P , 0.05) (Figures 4D–4F). Knockdown of up-regulated receptors was achieved by siRNA oligonucleotide transfection 48 hours before Hx challenge, and knockdown efficiency was verified by qPCR analysis (Figure 4G; Figures E7A–E7C). In silico analysis of transcriptional regulatory elements within the murine Tph1 promoter indicated

binding sites for a family of binding sites for the transcription factor activator protein 1 (AP1F/AP1), hypoxia-responsive element (HIFF/HRE), signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT6 transcription, representing potential Hx and/or PDGF responsive elements (Figure E8).

Figure 6. Imatinib treatment reduced right ventricular pressure (RVP), RV hypertrophy (RVH), and RV diastolic thickness beyond the protective effects of tryptophan hydroxylase 1 (Tph1) deletion. Wild-type (WT) and TPH12/2 mice (lack 5-hydroxytryptamine [5-HT] synthesis in the periphery) were maintained in normoxia (Nx) or hypoxia (Hx) conditions for 3 weeks. Subgroups were treated prophylactically twice daily with 100 mg/kg imatinib. (A) Mean RVP (mm Hg), (B) RVH, (C) RV thickness (mm) during diastole, and (D) systemic blood pressure (BP) in hypoxic WT (shaded bars) and TPH12/2 (solid bars) mice treated with imatinib or vehicle after vascular endothelial growth factor receptor inhibition. (E) Platelet-rich plasma 5-HT mean values in vehicle (SU) and imatinib (100 mg/kg, SU/Im) groups exposed to Nx or Hx. Data are mean 6 SEM for 10 animals per group. Statistical differences (*P , 0.05, **P , 0.01, **P , 0.001) are indicated by two-way analysis of variance. LV ¼ left ventricle; S ¼ septum.


85 Figure 7. Tryptophan hydroxylase 1 (Tph1)-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis. (Aa–Ad). The degree of muscularization is demonstrated by von Willebrand (vWF) (brown) and a-smooth muscle actin (a-SMA) (red) staining for identifying endothelium and vascular smooth muscle cells in small pulmonary arteries of wild-type (WT) and TPH12/2 hypoxic mice at 21 days’ treatment with imatinib. (Ae–Ah) Representative bright-field micrographs of proliferating cell nuclear antigen (PCNA)-stained lung tissue; arrows indicate positive proliferative cell staining. (Ai–Al) Representative bright-field micrographs of collagen fibers (PSR); arrows indicate perivascular localization (magnification 203). Morphometric analysis for specific staining quantification of (B) fully muscularized vessels, (C) PCNA, and (D) PSR. Results are expressed as mean 6 SEM (n ¼ 10). In C, the half-filled symbols are WT and the filled symbols are TPH12/2. Statistical differences (*P , 0.05, **P , 0.01, ***P , 0.001) are indicated by analysis of variance. (E) a-SMA and (F) collagen 3a1 (Col3a1) mRNA expression in lung homogenates were examined from hypoxic-challenged WT and TPH12/2 hypoxic animals receiving imatinib at 100 mg/kg/d, as compared with control mice. Quantitative changes in gene expression were analyzed by quantitative real-time polymerase chain reaction (DDCt method). Each bar shows mean 6 SEM of four animals per group.

Imatinib Treatment Decreased 5-HT and Tph1 Levels in BON Cells without Impacting TPH1 Enzymatic Activity

5-HT–producing BON cells were used to further investigate the novel mechanism of action for imatinib, demonstrating effective inhibition of serotonin release (half-maximal inhibitory concentration [IC50], 1.8 mM) (Figure 5A) and a complementary inhibition of Tph1 mRNA expression (Figure 5B). Furthermore, inhibition of PDGF signaling pathway in BON cells was demonstrated by reduced phosphorylation of PDGFR-b after PDGF stimulation (P , 0.05) (Figure 5C; Figure E8). Imatinibdependent effects on 5-HT production were not mediated by influencing enzyme activity, as demonstrated by an in vitro biochemical assay for human TPH1 (IC50 . 10 mM) compared with LP533401, a known TPH1 inhibitor (IC50, 120 nM) (Figure 5D). TPH1-Dependent Imatinib Efficacy in Collagen-mediated Perivascular Fibrosis

To better understand the consequences of TPH1-dependent imatinib efficacy on PAH pathology, we examined development of disease after 3 weeks of Hx/SU exposure in mice deficient in

Tph1 (Tph12/2) (24). As anticipated, increased 5-HT levels after Hx/SU exposure were ablated in Tph12/2 mice (Figure 6A). Imatinib exposure reduced all measures of PAH pathology observed in WT hypoxic mice (Figures 6B–6D, shaded bars). Hx-induced increases in RVP were partially attenuated in the Tph12/2 animals (Figure 6B), whereas RVH (Figure 6C) and RV diastolic thickness (Figure 6D) remained increased (solid bars). Treatment with imatinib reduced these measures beyond the protective effects of Tph1 deletion (P , 0.05) (Figures 6B–6D). Normoxic/SU5416 WT and Tph12/2 groups treated with imatinib did not display significant changes compared with vehicle-treated control mice (data not shown). Hx/SU-dependent increases in percentage of FM arterioles and PCNA proliferative vascular cells were equally attenuated on imatinib treatment (Figures 7Aa and 7Ab, 7Ae and 7Af, 7B, and 7C). The extensive changes in pulmonary vascular remodeling in response to Hx/SU were also attenuated in Tph12/2 mice, yet further decreased on imatinib treatment (Figures 7Ac and 7Ad, 7Ag and 7Ah, 7B, and 7C). However, imatinib did not significantly further impact the deposition of collagen in Hx/Tph12/2 mice, as demonstrated (Figures 7Ai–7Al and 7D). In parallel to

86


Figure 8. 5-Hydroxytryptamine (5-HT) levels in patients after 6 months of treatment with imatinib or placebo. Plasma 5-HT absolute levels in patients randomized to imatinib or placebo, stratified by baseline pulmonary vascular resistance (PVR) (Ai, Aii) >1,000 dynes s/cm5. (Bi, Bii) ,1,000 dynes s/cm5; ELISA data are mean 6 SEM for 5 to 12 patients per group and are expressed in ng/ml. 5-HT measured at baseline and 6 months after imatinib or placebo treatment. Red marks represent the mean change from baseline to study end in plasma 5-HT in patients randomized to imatinib or placebo. Statistical significance (*P , 0.05) is indicated by two-way analysis of variance.

normalization of vessel morphology, the increase in lung a-SMA and Col3a1 mRNA levels during Hx was considerably reduced in WT animals treated with imatinib (Figures 7E and 7F, shaded bars) (P , 0.05). A decrease in a-SMA expression in Tph12/2 mice was further impacted on imatinib treatment (Figure 7, solid bars) (P , 0.05). Interestingly, Col3a1 mRNA levels were ablated in Tph12/2 mice, with imatinib unable to further reduce mRNA levels (Figure 7F, solid bars). 5-HT Levels in Patients with PAH after 6 Months of Treatment with Imatinib or Placebo

Finally, we compared the effects of imatinib on the plasma levels of 5-HT in a randomized double-blind, placebo-controlled pilot study in patients with PAH who had not adequately improved with prostacyclin analogs (35). Although initially we observed no significant decreases in plasma 5-HT (Figure E8A), post hoc stratification of patients by baseline pulmonary vascular resistance (PVR) greater than or equal to 1,000 dyne s/cm5 revealed a statistically significant reduction in 5-HT after imatinib treatment compared with placebo (Figures 8Ai and 8Aii, 8Bi and 8Bii).

DISCUSSION We propose a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-b signaling.

VOL 187

2013

Our data reveal interplay between PDGF signaling and the 5-HT system within PAH, illustrating TPH1-dependent imatinib efficacy in collagen-mediated perivascular fibrosis. Furthermore, our study revealed a reduction in plasma 5-HT after imatinib treatment in patients with PAH with greater hemodynamic impairment from a published phase 2 clinical study, indicating that the novel mechanism of action we describe preclinically may translate to patients. Perturbation of a number of mechanisms, including pathways involving growth factors, inflammatory mediators, and metabolic signaling, may underlie the pathogenesis of PAH (36), yet how these pathways interact requires elucidation. Novel therapeutic agents, such as imatinib, inhibit several TKs associated with disease states, including BCR-ABL, c-Kit, and PDGF receptor a/b (4, 11). Imatinib efficacy has been demonstrated both preclinically and clinically in a subset of patients, although the precise mechanisms by which the drug exerts beneficial effects in PAH are poorly understood (25, 37). Further investigation of how imatinib works may help identify responder populations and enable the development of next-generation anti-remodeling therapies. To further understand imatinib mechanism of action in PAH, we investigated treatment during pathology development in the murine Hx/SU model of experimental PAH (27, 38). In Hx/SU mice, moderate to severe PAH developed within 21 days and imatinib treatment reduced all measures of pathology, as demonstrated by normalized hemodynamic values and improvement of cardiac output. Treatment did not affect SBP, suggesting selectivity of this approach for the abnormal pulmonary circulation and providing evidence for the absence of vasodilatory effects. As a consequence of reduced right heart load, muscular cardiac hypertrophy was decreased to baseline in hypoxic mice treated with imatinib. Structural changes observed in the above model more closely mimic human PAH in terms of (1) marked medial wall thickening, and (2) intimal proliferation (27). The proportion of FM vessels, occluded vessels, medial wall thickness, PCNApositive proliferating cells, and periadventitial collagen deposition increased under chronic hypoxic exposure were significantly reduced after imatinib treatment. In corroboration with previous studies (11, 36), we show that altered PDGF signaling plays an important role in the course of PAH, and that PDGFR antagonist imatinib is an effective treatment in the hypoxia/SU5416 experimental model of PAH. Furthermore, disease-associated biomarker analysis in mouse plasma revealed positive effects of imatinib treatment on vasoactive factors involved in the development of medial thickening as well as inflammatory cell recruitment (36, 39, 40). Of the biomarkers analyzed, the most intriguing change observed with imatinib treatment was the inhibition of elevated 5-HT. Association of the 5-HT system with PAH has been well established, from serotoninergic appetite suppressant drugs increasing the risk of disease to later demonstration of direct pathologic roles in both vasoconstriction and remodeling of the pulmonary vasculature and microthrombosis (18, 41, 42). PAEC-derived 5-HT acts as a potent mitogenic factor on SMCs in pulmonary vessels (19, 21) and has been shown to transactivate PDGFRb in PASMCs leading to SMC proliferation and migration (25). These findings are consistent with the observation that 5-HT and TPH1 levels are increased in lungs and endothelial cells from patients with PAH (4, 16, 22, 23). Furthermore, the importance of 5-HT signaling for fibrotic diseases is supported by recent studies in which 5-HTR2A/2B receptor antagonist (terguride) was found to reduce RVP, SMCs proliferation, and collagen accumulation (43). To validate a potential pharmacological effect of imatinib on 5-HT, we examined several components of the signaling pathway


Figure 9. Schematic of the proposed signaling events in hypoxiainduced pulmonary arterial hypertension (PAH) and the impact of imatinib on the 5-hydroxytryptamine (5-HT) system. Signaling cascade initiated by hypoxia (Hx) exposure was substantially diminished after imatinib treatment of lung and HPAECs. Platelet-derived growth factor (PDGF)-BB alone may induce tryptophan hydroxylase 1 (Tph1) expression and subsequent 5-HT release, as STATs and transcription factor activator protein 1 (AP1F/AP1)-binding sites were found in TPH1 promoter region. TPH1 synthesis and subsequent 5-HT release from pulmonary artery endothelial cells (PAECs) may act as a mitogen on pulmonary artery smooth muscle cells and adjacent fibroblasts, contributing to pulmonary vascular remodeling, extracellular matrix (ECM) deposition, and subsequent onset of PAH. Induction of pathways and/ or cellular events are symbolized by black arrows (solid lines indicate proven mechanism; dashed lines indicate postulated mechanisms). Red lines and arrows indicate the actions of imatinib in this model. Col3a1 ¼ collagen 1a1; SMA ¼ smooth muscle actin; STAT ¼ signal transducer and activator of transcription.

in lung tissue. In accordance with the elevated 5-HT after chronic hypoxic exposure, increased expression of TPH1, 5-HT receptors, and 5-HT transporter was observed in lung tissue. Imatinib abolished the later increases in TPH1 but did not affect the early peaks in 5-HT receptors and transporter levels. The compound did not reduce brain Tph1 and Tph2 levels under chronic hypoxic exposure and therefore shows no consequent central nervous system liabilities (44). Using in vitro functional studies, we identified Pdgfr-b signaling but not Pdgfr-a or c-Kit as required for Tph1 downregulation by imatinib. The regulatory role of Pdgfr-b on Tph1 expression was further investigated in silico, where HIFF/HRE, STAT3, STAT6, and AP1F/AP1-binding sites were identified in the Tph1 promoter region, representing potential Hx and PDGF responsive elements for direct transcriptional regulation. It has previously been demonstrated that Hif-1 binds directly to the Tph-1 promoter via the HRE conserved site to drive 5-HT production in hypoxic conditions in Caenorhabditis elegans (45). The pattern of PDGF/Tph1 interaction was confirmed in BON cells—a carcinoid line that constitutively produces high levels of 5-HT via TPH1 (30). Imatinib treatment lowered 5-HT levels in BON cell lysates in a dose-dependent manner. PDGFR-b pathway activation in untreated BON cells implies the expression and

87

release of PDGF into the culture medium, which may act in an autocrine or paracrine fashion, thereby explaining the imatinib inhibitory effects. Similar, Yao and colleagues have used treated BON-1 cells as an in vitro model to explore activity and efficacy of imatinib in patients with carcinoid tumors and showed clinical improvement (46). Interestingly, imatinib-dependent effects on 5-HT production were mediated via inhibition of TPH1 expression and not by influencing enzyme activity. To better understand the consequences of TPH1-dependent imatinib efficacy, we treated Tph12/2 mice in the Hx/SU model. As anticipated, peripheral 5-HT levels were absent in Tph12/2 mice. In corroboration with previous publication (24), Tph12/2 mice are protected against Hx-induced increases in RVP but still develop RVH. This suggests that in mice, Hx can directly induce RVH, independent of peripheral 5-HT or increases in RVP. For example, it has been reported previously that serotonin transporter (SERT)1 mice show elevated RVP in the absence of RVH (47). Treatment with imatinib reduced all measures of PAH pathology, beyond the protective effects of Tph1 deletion. Unexpectedly, imatinib did not significantly further impact the deposition of collagen and Col3a1 levels in hypoxic Tph12/2 mice. Recent reports indicate both c-ABL–dependent and -independent reduction in SMCs proliferation by imatinib via the PI3K/Akt–protein kinase B pathway (11). We conclude that the TPH1-inhibitory capacity of imatinib is most likely the predominant mode of action involved in the beneficial antifibrotic effects in this PAH model. Inhibition of various 5-HT pathway elements have also been shown to impact fibrosis within in vitro and in vivo models of PAH (22, 23, 26, 43, 47–52). Data from a phase 2 study suggest that imatinib may be efficacious as an adjunct therapy in patients with inadequate response to at least one other PAHtargeted treatment (34). Greatly improved responsiveness was observed in a subset of patients whose baseline PVR was greater than 1,000 dynes. Similarly, our study revealed a statistically significant reduction in plasma 5-HT after imatinib treatment in the same group of patients, indicating that the novel mechanism of action we describe preclinically may translate to patients. However, given its limited nature (sample size and preparation), it may be worthwhile to assess the potential role of 5-HT as a biomarker in further studies. In summary, we propose a pathologic link between PDGFR-b phosphorylation and subsequent enhancement of TPH1 expression in PAH (Figure 9). This signaling cascade initiated by hypoxic exposure was substantially diminished after imatinib treatment of lung and HPAECs. PDGF-BB alone may induce TPH1 expression and subsequent 5-HT release, as STATs and AP1F/AP1-binding sites were found in the Tph1 promoter region. TPH1 synthesis and subsequent 5-HT release from PAECs acts as a mitogen on PASMCs and adjacent fibroblasts, contributing to pulmonary vascular remodeling, extracellular matrix deposition, and subsequent onset of PAH. Inhibition of PDGFR signaling via imatinib may sever the link to TPH1 expression and 5-HT production, thus reducing the vascular remodeling and profibrotic processes characteristic of preclinical and clinical PAH. In conclusion, we report for the first time a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-b signaling. Our data reveal interplay between PDGF signaling and the 5-HT system within PAH, demonstrating TPH1-dependent imatinib efficacy in collagenmediated mechanisms of perivascular fibrosis. Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank the members of the Laboratory Animal Services (Andy Nicholls, Barrie Sandells, Dave Bateman, Michael George, and Sarah Lane) and the members of the Histology Department, led by Dr. Paul Whittaker, for excellent technical assistance.

88


References 1. Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, McMurtry IF, Stenmark KR, Thistlethwaite PA, Weissmann N, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54:S20–S31. 2. Drake JI, Bogaard HJ, Mizuno S, Clifton B, Xie B, Gao Y, Dumur CI, Fawcett P, Voelkel NF, Natarajan R. Molecular signature of a right heart failure program in chronic severe pulmonary hypertension. Am J Respir Cell Mol Biol 2011;45:1239–1247. 3. O’Callaghan DS, Savale L, Montani D, Jais X, Sitbon O, Simonneau G, Humbert M. Treatment of pulmonary arterial hypertension with targeted therapies. Nat Rev Cardiol. 2011;8:526–538. 4. Schermuly RT, Ghofrani HA, Wilkins MR, Grimminger F. Mechanisms of disease: pulmonary arterial hypertension. Nat.Rev.Cardiol. 2011;8: 443–455. 5. Fares WH, Trow TK. Targeted approaches to the treatment of pulmonary hypertension. Ther Adv Respir Dis 2012;6:147–159. 6. Chhina MK, Nargues W, Grant GM, Nathan SD. Evaluation of imatinib mesylate in the treatment of pulmonary arterial hypertension. Future Cardiol 2010;6:19–35. 7. Perros F, Montani D, Dorfmuller P, Durand-Gasselin I, Tcherakian C, Le Pavec J, Mazmanian M, Fadel E, Mussot S, Mercier O, et al. Platelet-derived growth factor expression and function in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2008; 178:81–88. 8. ten Freyhaus H, Dumitrescu D, Berghausen E, Vantler M, Caglayan E, Rosenkranz S. Imatinib mesylate for the treatment of pulmonary arterial hypertension. Expert Opin Investig Drugs 2012;21:119–134. 9. Ogawa A, Firth AL, Smith KA, Maliakal MV, Yuan JXJ. PDGF enhances store-operated Ca21 entry by upregulating STIM1/Orai1 via activation of Akt/mTOR in human pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 2012;302:C405–C411. 10. Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones PL, Maitland ML, Michelakis ED, Morrell NW, et al. Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 2009;54:S10–S19. 11. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811–2821. 12. Balasubramaniam V, Le Cras TD, Ivy DD, Grover TR, Kinsella JP, Abman SH. 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–L833. 13. Manley PW, Stiefl N, Cowan-Jacob SW, Kaufman S, Mestan J, Wartmann M, Wiesmann M, Woodman R, Gallagher N. Structural resemblances and comparisons of the relative pharmacological properties of imatinib and nilotinib. Bioorg Med Chem 2010;18:6977– 6986. 14. Ruiz PA, Jarai G. Collagen I induces discoidin domain receptor (DDR) 1 expression through DDR2 and a JAK2–ERK1/2-mediated mechanism in primary human lung fibroblasts. J Biol Chem 2011;286:12912– 12923. 15. Day E, Waters B, Spiegel K, Alnadaf T, Manley PW, Buchdunger E, Walker C, Jarai G. Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib. Eur J Pharmacol 2008;599:44–53. 16. MacLean MR, Dempsie Y. The serotonin hypothesis of pulmonary hypertension revisited. Adv Exp Med Biol 2010;661:309–322. 17. MacLean MR. Pulmonary hypertension, anorexigens and 5-HT: pharmacological synergism in action? Trends Pharmacol Sci 1999;20:490–495. 18. MacLean MR, Herve P, Eddahibi S, Adnot S. 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br J Pharmacol 2000;131:161–168. 19. Guignabert C, Raffestin B, Benferhat R, Raoul W, Zadigue P, Rideau D, Hamon M, Adnot S, Eddahibi S. Serotonin transporter inhibition prevents and reverses monocrotaline-induced pulmonary hypertension in rats. Circulation 2005;111:2812–2819. 20. Jaffre F, Bonnin P, Callebert J, Debbabi H, Setola V, Doly S, Monassier L, Mettauer B, Blaxall BC, Launay JM, et al. Serotonin and angiotensin receptors in cardiac fibroblasts coregulate adrenergic-dependent cardiac hypertrophy. Circ Res 2009;104:113–123.

VOL 187

2013

21. Eddahibi S, Guignabert C, Barlier-Mur AM, Dewachter L, Fadel E, Dartevelle P, Humbert M, Simonneau G, Hanoun N, Saurini F, et al. Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: critical role for serotonin-induced smooth muscle hyperplasia. Circulation 2006;113:1857–1864. 22. Eddahibi S, Adnot S. Serotonin and pulmonary arterial hypertension. Rev Mal Respir 2006;23:S45–S51. 23. Izikki M, Hanoun N, Marcos E, Savale L, Barlier-Mur AM, Saurini F, Eddahibi S, Hamon M, Adnot S. Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice. Am J Physiol Lung Cell Mol Physiol 2007;293:L1045–L1052. 24. Morecroft I, Dempsie Y, Bader M, Walther DJ, Kotnik K, Loughlin L, Nilsen M, MacLean MR. Effect of tryptophan hydroxylase 1 deficiency on the development of hypoxia-induced pulmonary hypertension. Hypertension 2007;49:232–236. 25. Ren W, Watts SW, Fanburg BL. Serotonin transporter interacts with the PDGFbeta receptor in PDGF-BB-induced signaling and mitogenesis in pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2011;300:L486–L497. 26. Liu Y, Li M, Warburton RR, Hill NS, Fanburg BL. The 5-HT transporter transactivates the PDGFbeta receptor in pulmonary artery smooth muscle cells. FASEB J 2007;21:2725–2734. 27. Ciuclan L, Bonneau O, Hussey M, Duggan N, Holmes AM, Good R, Stringer R, Jones P, Morrell NW, Jarai G, et al. A novel murine model of severe pulmonary arterial hypertension. Am J Respir Crit Care Med 2011;184:1171–1182. 28. Ciuclan L, Bonneau O, Hussey M, Burton V, Good R, Duggan N, Thomas M. The roles of TPH1, serotonin and PDGF within a novel murine model of pulmonary arterial hypertension [abstract]. 2011. Am J Respir Crit Care Med 2011;183:A4962. 29. Dooley S, Hamzavi J, Ciuclan L, Godoy P, Ilkavets I, Ehnert S, Ueberham E, Gebhardt R, Kanzler S, Geier A, et al. Hepatocytespecific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology 2008;135: 642–659. 30. Tran VS, Marion-Audibert AM, Karatekin E, Huet S, Cribier S, Guillaumie K, Chapuis C, Desnos C, Darchen F, Henry JP. Serotonin secretion by human carcinoid BON cells. Ann N Y Acad Sci 2004; 1014:179–188. 31. Burton VJ, Ciuclan LI, Holmes AM, Rodman DM, Walker C, Budd DC. Bone morphogenetic protein receptor-II regulates pulmonary artery endothelial cell barrier function. Blood 2011;117:333–341. 32. Quandt K, Frech K, Karas H, Wingender E, Werner T. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995;23:4878–4884. 33. Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 2005; 21:2933–2942. 34. Ghofrani HA, Seeger W, Grimminger F. Imatinib for the treatment of pulmonary arterial hypertension. N Engl J Med 2005;353:1412–1413. 35. Ghofrani HA, Morrell NW, Hoeper MM, Olschewski H, Peacock AJ, Barst RJ, Shapiro S, Golpon H, Toshner M, Grimminger F, et al. Imatinib in pulmonary arterial hypertension patients with inadequate response to established therapy. Am J Respir Crit Care Med 2010;182: 1171–1177. 36. Dahal BK, Heuchel R, Pullamsetti SS, Wilhelm J, Ghofrani HA, Weissmann N, Seeger W, Grimminger F, Schermuly RT. Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptor-b. Pulm Circ 2011;1:259–268. 37. Humbert M, Montani D, Perros F, Dorfmuller P, Adnot S, Eddahibi S. Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension. Vascul Pharmacol 2008;49:113–118. 38. Agarwal A, Fleischman AG, Petersen CL, Loriaux MM, Woltjer R, Druker BJ, Deininger MW. Effects of plerixafor (AMD3100) in combination with tyrosine kinase inhibition in a murine model of CML. Blood 2010;116:1387–1388. 39. Heresi GA. Clinical perspective: biomarkers in pulmonary arterial hypertension. Int J Clin Pract 2011;65:5–7.

Ciuclan, Hussey, Burton, et al.: Interplay between PDGF and 5-HT Pathways within PAH 40. Koyanagi M, Egashira K, Kitamoto S, Ni WH, Shimokawa H, Takeya M, Yoshimura T, Takeshita A. Role of monocyte chemoattractant protein-1 in cardiovascular remodeling induced by chronic blockade of nitric oxide synthesis. Circulation 2000;102:2243–2248. 41. MacLean MR, Dempsie Y. Serotonin and pulmonary hypertension– from bench to bedside? Curr Opin Pharmacol 2009;9:281–286. 42. McLaughlin VV, Davis M, Cornwell W. Pulmonary arterial hypertension. Curr Probl Cardiol 2011;36:461–517. 43. Dumitrascu R, Kulcke C, Konigshoff M, Kouri F, Yang X, Morrell N, Ghofrani HA, Weissmann N, Reiter R, Seeger W, et al. Terguride ameliorates monocrotaline-induced pulmonary hypertension in rats. Eur Respir J 2011;37:1104–1118. 44. Takayama N, Sato N, O’Brien SG, Ikeda Y, Okamoto SI. Imatinib mesylate has limited activity against the central nervous system involvement of Philadelphia chromosome-positive acute lymphoblastic leukaemia due to poor penetration into cerebrospinal fluid. Br J Haematol 2002; 119:106–108. 45. Pocock R, Hobert O. Oxygen levels affect axon guidance and neuronal migration in Caenorhabditis elegans. Nat Neurosci 2008;11:894–900. 46. Yao JC, Zhang JX, Rashid A, Yeung SCJ, Szklaruk J, Hess K, Xie KP, Ellis L, Abbruzzese JL, Ajani J. Clinical and in vitro studies of imatinib in advanced carcinoid tumors. Clin Cancer Res 2007;13: 234–240.

89

47. MacLean MR, Deuchar GA, Hicks MN, Morecroft I, Shen S, Sheward J, Colston J, Loughlin L, Nilsen M, Dempsie Y, et al. Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation 2004;109:2150–2155. 48. Konigshoff M, Dumitrascu R, Udalov S, Amarie OV, Reiter R, Grimminger F, Seeger W, Schermuly RT, Eickelberg O. Increased expression of 5-hydroxytryptamine(2A/B) receptors in idiopathic pulmonary fibrosis: a rationale for therapeutic intervention. Thorax 2010;65:949–955. 49. Maclean M, Morecroft I, Dempsie Y. The serotonin transporter in pulmonary arterial hypertension. Fundam Clin Pharmacol 2008;22:24. 50. Mair KM, MacLean MR, Morecroft I, Dempsie Y, Palmer TM. Novel interactions between the 5-HT transporter, 5-HT(1B) receptors and Rho kinase in vivo and in pulmonary fibroblasts. Br J Pharmacol 2008;155:606–616. 51. White K, Loughlin L, Maqbool Z, Nilsen M, McClure J, Dempsie Y, Baker AH, MacLean MR. Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH. Physiol Genomics 2011;43:417–437. 52. White K, Dempsie Y, Nilsen M, Wright AF, Loughlin L, MacLean MR. The serotonin transporter, gender, and 17 beta oestradiol in the development of pulmonary arterial hypertension. Cardiovasc Res 2011; 90:373–382.