Published ahead of print on April 17, 2008, doi:10.1164/rccm.200707-1037OC
American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 81-88, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200707-1037OC
Platelet-derived Growth Factor Expression and Function in Idiopathic Pulmonary Arterial Hypertension
Frédéric Perros1,2,3,
David Montani1,2,
Peter Dorfmüller1,2,
Ingrid Durand-Gasselin2,
Colas Tcherakian1,2,
Jérôme Le Pavec1,
Michel Mazmanian3,
Elie Fadel3,
Sacha Mussot3,
Olaf Mercier3,
Philippe Hervé3,
Dominique Emilie2,
Saadia Eddahibi4,
Gérald Simonneau1,
Rogério Souza1,2 and
Marc Humbert1,2
1 Université Paris-Sud 11, UPRES EA 2705, Centre National de Référence de l'Hypertension Artérielle Pulmonaire, Service de Pneumologie et Réanimation Respiratoire, Institut Paris-Sud Cytokines, Hôpital Antoine-Béclère, Assistance Publique Hôpitaux de Paris, Clamart, France; 2 INSERM U764, Clamart, France; 3 UPRES EA 2705, Laboratoire de Chirurgie Expérimentale, Centre Chirurgical Marie Lannelongue, Université Paris-Sud 11, Le Plessis Robinson, France; and 4 INSERM U841 and Département de Physiologie Explorations Fonctionnelles, Hôpital Henri-Mondor, Assistance Publique Hôpitaux de Paris, Créteil, France
Correspondence and requests for reprints should be addressed to Marc Humbert, M.D., Ph.D., Service de Pneumologie et Réanimation Respiratoire, Hôpital Antoine-Béclère, 157 rue de la Porte de Trivaux, 92140 Clamart, France. E-mail: marc.humbert{at}abc.aphp.fr
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ABSTRACT
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Rationale: Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries.
Objectives: To analyze the expression and pathogenic role of PDGF in idiopathic PAH.
Methods: PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription–polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH. Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-β. The effects of imatinib on PDGF-B–induced proliferation and chemotaxis were tested on human PASMCs.
Measurements and Main Results: PDGF-A, PDGF-B, PDGFR- , and PDGFR-β mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-β in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR- and PDGFR-β mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-β. PDGF-BB–induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis.
Conclusions: PDGF may play an important role in human PAH. Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.
Key Words: imatinib • pulmonary arterial hypertension • platelet-derived growth factor • remodeling • smooth muscle cells
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AT A GLANCE COMMENTARY
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Scientific Knowledge on the Subject
Platelet-derived growth factor (PDGF) plays an important part in the progression of experimental pulmonary hypertension, but its role in human pulmonary arterial hypertension is only partly understood.
What This Study Adds to the Field
Expression of PDGF and PDGF receptors is increased in the pulmonary arteries of patients with pulmonary arterial hypertension. PDGF induces proliferation and migration of human pulmonary artery smooth muscle cells, which is inhibited by imatinib, a PDGF receptor inhibitor.
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Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and ultimately death (1). Remodeling of small pulmonary arteries represents the main pathologic finding related to PAH with marked proliferation of pulmonary artery smooth muscle cells (PASMCs), resulting in the obstruction of resistance pulmonary arteries (2, 3). Several mechanisms have been described in the pathogenesis of PAH, including those related to current therapeutic targets such as endothelin-1, prostacyclin, and nitric oxide (1, 3). In recent years, recognition of germline mutations of genes coding for receptor members of the transforming growth factor-β superfamily (4) in heritable PAH has emphasized the potential role of growth factors in the development of PAH. Among them, platelet-derived growth factor (PDGF) has been identified as a novel possible therapeutic target in PAH (5, 6).
Active PDGF is built up by polypeptides (A and B chain) that form homo- or heterodimers and stimulate - and β-cell surface receptors (7). Recently, two additional PDGF genes have been identified, encoding PDGF-C and PDGF-D polypeptides (8). PDGF is synthesized by many different cell types, including smooth muscle cells (SMCs), endothelial cells, and macrophages (7). PDGF has the ability to induce the proliferation and migration of SMCs and fibroblasts, and it has been proposed as a key mediator in the progression of several fibroproliferative disorders such as atherosclerosis, lung fibrosis, and pulmonary hypertension (6–8). It can also induce the contraction of rat aorta strips in vitro (9).
Novel therapeutic agents such as imatinib mesylate (Gleevec; Novartis, Horsham, UK) inhibit several tyrosine kinases associated with disease states, including BCR-ABL (breakpoint cluster region-Abelson) in patients with chronic myelogenous leukemia, c-kit in patients with gastrointestinal stromal tumors, and PDGF receptors and β in patients with certain myeloproliferative disorders and dermatofibrosarcoma protuberans, respectively (10, 11). Imatinib has been demonstrated to reverse pulmonary vascular remodeling in animal models of pulmonary hypertension (6) and a few cases of clinical and hemodynamic improvements have also been reported in human PAH (12–14). Due to the lack of comprehensive human data, we performed a complete analysis of the pathogenic role of PDGF in human PAH. We first confirmed increased expression of PDGF and PDGF receptors by means of real-time reverse transcription–polymerase chain reaction (RT-PCR) performed on laser capture microdissected pulmonary arteries from lung transplanted patients displaying severe idiopathic PAH. Western blot analysis showed increased PDGFR-β protein expression in PAH lungs, as compared with controls. Immunohistochemistry techniques were then used to localize PDGF-A and PDGF-B and PDGFR- and PDGFR-β proteins in PAH lungs. The effect of imatinib on PDGFR phosphorylation in cultured PASMCs was studied by Western blot, and PASMC apoptosis was analyzed by fluorometric detection of caspase 3 and 7 activity. Last, we analyzed the effects of imatinib on PDGF-B–induced proliferation and chemotaxis on control and PAH PASMCs. Our data support the concept that PDGF is overproduced within the pulmonary artery wall of patients with PAH and promotes pulmonary arterial remodeling.
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METHODS
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Lung Samples and PASMC Cultures
Human lung specimens were obtained at the time of lung transplantation (PAH, n = 13) or from tissue obtained during lobectomy or pneumonectomy for localized lung cancer (controls, n = 8), then snap frozen or paraffin embedded, as previously described (15). PASMCs were cultured from the same explants as previously described (16). In brief, arteries (diameter: 5–10 mm) were kept in Dulbecco's modified Eagle medium (DMEM) at 4°C before their intimal cell layer and residual adventitial tissue were stripped off using forceps. The dissected media of the vessels was then cut into small pieces (3–5 mm), which were transferred into cell culture flasks. To allow the PASMCs to grow out, the vessel tissues were incubated in DMEM supplemented with 20% fetal calf serum (FCS), 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). After 2 weeks of incubation, the PASMCs collected in the culture medium and the vessel tissues were transferred into new cell culture flasks. This study was approved by the local ethics committee and patients agreed to contribute to the study.
Laser Capture Microdissection of Pulmonary Arteries, cDNA Preparation, and RT-PCR
Small pulmonary arteries (100–200 µm) were captured using the ASLMD laser microdissection microscope (Leica, Rueil-Malmaison, France). RNA was extracted from microdissected pulmonary arteries with a PicoPure RNA isolation kit (Arcturus, Mountain View, CA) and then eluted from silicate columns and reverse-transcribed using Sensiscript Reverse Transcription kit (Qiagen, Courtaboeuf, France). Constitutively expressed β-actin was selected as an internal housekeeping gene control for the comparative cycle threshold (CT) method for the relative quantification of PDGF-A and PDGF-B, and PDGF receptor and β. PDGF-A and PDGF-B, PDGF receptor and β, and β-actin expressions were quantified by RT-PCR with TaqMan gene expression assays (assay ID number in brackets) (β-actin [Hs99999903_m1], PDGF-A [Hs00234994_m1], PDGFR- [Hs00183486_m1], PDGF-B [Hs00234042_m1], PDGFR-β [Hs00182163_m1]), and TaqMan Universal PCR Master Mix performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France).
Western Blot
Lung tissue samples from 10 control subjects and 10 patients with PAH were homogenized in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1.5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 3% glycerol, 0.2 mM orthovanadate, and protease inhibitor cocktail (aprotine, leupeptine, and PefaBloc [Roche, Meylan, France]). Lysates were normalized and separated on 8% polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, the membranes were probed with rabbit anti–PDGFR-β (1µg/ml, cat. no. sc432) or anti–phospho-PDGFR-β (1µg/ml, cat. no. sc12909-R) and anti–actin-β loading control (0.2 µg/ml, cat. no. sc-1615) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Bound antibodies were detected by chemiluminescence with the use of an enhanced chemiluminescence (ECL) detection system (Millipore, Paris, France) and quantified by densitometry.
Immunohistochemistry
Immunohistochemistry was either performed on 8-µm-thick sections of frozen tissue (PDGF-AA and PDGF-BB, and PDGFR- and PDGFR-β), or on 3-µm-thick sections of paraffin embedded tissue (PDGF-BB, phosphorylated PDGFR-β, proliferating cell nuclear antigen [PCNA]). After routine preparation and microwave unmasking, slides were processed with rabbit anti–PDGF-BB (1 µg/ml, ab15499; Abcam, Paris, France), PDGFR-β (2 µg/ml, sc-432), PDGF-AA (2 µg/ml, sc-128), PDGFR- (2 µg/ml, sc-338), phospho-PDGFR-β (2 µg/ml, sc-12909-R), and PCNA (2 µg/ml, sc-7907) (all sc catalog numbers are from Santa Cruz Biotechnology, Inc.). According to the manufacturer's recommendations, the Envision kit (K4065; Dako, Trappes, France) was used for primary antibody detection. Controls used for these antibodies included omission of the primary antibody and substitution of the primary antibody by rabbit IgG.
PASMC Proliferation Assay
PASMCs were serum-starved for 48 hours (0.2% FCS), then incubated with PDGF-BB (10 and 50 ng/ml), epithelial growth factor (EGF) or basic fibroblast growth factor (bFGF) (50 ng/ml; R&D Systems Europe, Lille, France), with or without 5 µM imatinib (STI571; Novartis, Basel, Switzerland) for 24 hours with [3H]thymidine (Amersham France, Les Ulis, France), and cell proliferation was detected by thymidine incorporation (16).
For cell counting, PASMCs were allowed to adhere overnight on Labtek 8 chamber slides (Nunc, Wiesbaden, Germany), then were treated with the above conditions during 48 days. The chambers were then removed, the slides washed in phosphate-buffered saline, fixed in acetone, and stained with 4'-6-diamidino-2-phenylindole (DAPI). The DAPI-stained cells were visualized under a Nikon eclipse 80i fluorescent microscope and the DAPI-positive cells were automatically counted (NIS Element BR2.30 software) (Nikon France, Champigny sur Marne, France).
Apoptosis Assay
The apoptosis was quantified by the measurement of caspase 3 and 7 activity in PASMCs. Caspase activity was detected within whole living cells using Immunochemistry Technologies (ICT)'s Magic Red substrate-based MR-caspase assay kit according to the manufacturer's instructions (Serotec, Dusseldorf, Germany). Hydrogen peroxide 1 mM was chosen as a positive control for SMC apoptosis (17).
PASMC Migration Assay
PASMC migration was evaluated by the transwell assay. Trypsinized PASMCs were transferred into the upper chambers of 8-µm-pore transwell plates (VWR, Fontenay-sous-Bois, France). PDGF-BB (10 or 100 ng/ml), EGF, or bFGF (50 ng/ml; R&D Systems Europe, Lille, France), with or without 5 µM imatinib, was added to the lower chamber. After 24 hours at 37°C, migration was quantified by counting cells in the bottom of the membrane stained with DiffQuick (Dade Behring S.A., Paris la Defense, France). The number of cells on the lower surface of filter was counted by light microscopy under high-power field (x200). Eight fields were counted in each of three different experiments.
Statistical Evaluation
Quantitative variables were presented as means ± SD. Between groups, comparisons were made with Student's t test; multiple group comparisons were performed with analysis of variance and the least significant difference method as a post hoc analysis. P values less than 0.05 were considered to reflect statistical significance.
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RESULTS
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PDGF and PDGF-Receptor mRNA Expression in Microdissected Pulmonary Arteries
PDGF-A, PDGF-B, PDGFR-, and PDGFR-β mRNA expression was increased in microdissected small pulmonary arteries from patients displaying severe PAH, as compared with control subjects (P < 0.01 for PDGF-B, PDGFR-, PDGFR-β, and P = 0.09 for PDGF-A; Figure 1).
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Figure 1. Platelet-derived growth factor (PDGF) and PDGF receptor (PDGFR) expression in microdissected pulmonary arteries from patients with severe pulmonary arterial hypertension (PAH) and from control subjects.
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PDGF-Receptor Protein Expression in Total Lung
Western blot analysis revealed a significant increase in protein expression of PDGFR-β in PAH lungs compared with control lungs. PDGFR-β protein expression normalized to β-actin was 0.89 ± 0.43 in control lungs and 1.64 ± 0.77 in PAH lungs (P = 0.01) (Figure 2).
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Figure 2. Platelet-derived growth factor receptor (PDGFR)-β protein expression in total lung of control subjects and patients with pulmonary arterial hypertension (PAH).
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Immunohistochemistry
In small pulmonary arteries with constrictive or plexiform remodeling, PDGF-A and PDGF-B expression was mainly localized to smooth muscle and endothelial cells (Figures 3A, 3C, and 4A). In addition, perivascular inflammatory infiltrates, when present, showed intensive staining (Figures 3A, 5A, and 5B). PDGFR- and PDGFR-β were mainly expressed in SMCs and to a lesser extent in endothelial cells (Figures 3B and 3D). The phosphorylated form of PDGFR-β was detected in both smooth muscle and endothelial cells, depending on the predominant proliferating vascular compartment of constrictive and plexiform lesions (Figures 4C–4D). Proliferating cells were identified with PCNA and corresponded to endothelial and SMCs within pulmonary vascular lesions (Figure 4B).
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Figure 3. Immunohistochemistry: localization of platelet-derived growth factor (PDGF)-A, PDGF-B and their receptors in pulmonary arterial lesions of patients with pulmonary arterial hypertension. (A) Constrictive lesion of a pulmonary artery with perivascular cells expressing PDGF-A (arrows). (B) Constrictive lesion of a pulmonary artery: PDGFR- expression is mainly detected within the muscular medial layer displaying hypertrophy. (C) Plexiform lesion displaying PDGF-B endothelial cell expression (arrow). (D) Same lesion stained with the anti–PDGFR-β. Note positive smooth muscle cells within the preserved medial layer (arrow) and the remodeled intima (asterisk).
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Figure 4. Platelet-derived growth factor (PDGF)-B and phosphorylated receptor expression in pulmonary arterial lesions of patients with pulmonary arterial hypertension. (A) Plexiform lesion displaying PDGF-B endothelial cell expression within characteristic endothelium-lined channels (arrows). (B) Same lesion highlighting the proliferating cells through PCNA (proliferating cell nuclear antigen) staining. (C) A larger pulmonary artery with medial hypertrophy expresses the phophorylated form of PDGFR-β within smooth muscle cells (bold arrow) and endothelial cells (thin arrows). (D) Plexiform lesion with multiple intraluminal endothelium-lined channels expressing the phophorylated form of PDGFR-β within endothelial cells.
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Figure 5. Platelet-derived growth factor (PDGF)-B expression in pulmonary arterial lesions of patients with pulmonary arterial hypertension. (A) Small plexiform lesion of a pulmonary artery. Note the perivascular lymphoid infiltrate (arrows). Hematoxylin–eosin–saffron staining. (B) Same lesion showing PDGF-B expression within lumen-occluding smooth muscle and endothelial cells. Inflammatory cells to the upper right show strong staining.
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Effect of PDGF-BB and Imatinib on PDGFR-β Phosphorylation
PDGF-BB 10 ng/ml significantly increased phosphorylated PDGFR-β protein expression compared with control conditions (P < 0.05). Imatinib given at the same time as PDGF-BB partially decreased PASMC PDGFR-β phosphorylation induced by 10 minutes of PDGF-BB stimulation (P = 0.06 compared with PDGF-BB 10 ng/ml; Figure 6). Imatinib given 90 minutes before adding PDGF-BB completely blocked PDGFR-β phosphorylation (P < 0.01 compared with PDGF-BB 10 ng/ml; Figure 6).
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Figure 6. Effect of platelet-derived growth factor (PDGF)-BB and imatinib (STI571) on PDGFR-β phosphorylation. Western blot analysis was used to assess expression of phosphorylated PDGFR-β in pulmonary artery smooth muscle cells treated with PDGF-BB and imatinib. Phosphorylated PDGFR-β protein expression was normalized to β-actin. Immunoblots are representative of phosphorylated PDGFR-β expression. PDGF-BB increased phosphorylated PDGFR-β protein expression compared with control conditions. Imatinib given at the same time as PDGF-BB partially decreased PDGFR-β phosphorylation induced by 10 minutes of PDGF-BB stimulation, whereas imatinib given 90 minutes before adding PDGF-BB (imatinib pretreatment) completely blocked PDGFR-β phosphorylation. *P < 0.05 versus control; #P < 0.01 versus pretreatment with imatinib.
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Effect of Imatinib on Cultured PASMC Proliferation
PASMC proliferation, induced by PDGF-BB 10 ng/ml, was inhibited by imatinib (5 µM), as demonstrated by [3H]thymidine incorporation assay. To determine the selectivity of STI571 on PDGF-induced PASMC proliferation, other growth factors (EGF or bFGF, both at 50 ng/ml) were used to stimulate PASMCs. Although imatinib demonstrated a significant inhibition of PDGF-BB–induced proliferation (P < 0.0001), proliferation stimulated by EGF or bFGF was unaffected (Figure 7).
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Figure 7. Effect of imatinib on platelet-derived growth factor (PDGF)-B–, epithelial growth factor (EGF)-, and basic fibroblast growth factor (bFGF)-induced pulmonary artery smooth muscle cell (PASMC) proliferation (tritiated thymidine incorporation assay). Although imatinib demonstrated a significant inhibition of PDGF-BB–induced proliferation (P < 0.0001), proliferation stimulated by EGF or bFGF was unaffected. *P < 0.01 versus control; **P < 0.001 versus control; ***P < 0.0001 versus control; #P < 0.0001 versus condition without imatinib.
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We confirmed the inhibitory effect of imatinib on PDGF-BB–induced PASMC proliferation by cell counting (Figure 8). PDGF-BB (50 ng/ml) induced PASMC proliferation similar to PDGF-BB (10 ng/ml) (155.6 ± 24.9% and 173.7 ± 15.3% of control conditions, P = 0.21). Imatinib inhibited significantly the proliferation induced by PDGF-BB 10 ng/ml or 50 ng/ml (P = 0.001 and P < 0.001, respectively).
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Figure 8. Effects of platelet-derived growth factor (PDGF)-BB and imatinib (STI571) on pulmonary artery smooth muscle cell (PASMC) proliferation assessed by cell counting. PASMC proliferation was assessed by cell counting (number of 4'-6-diamidino-2-phenylindole [DAPI]–positive cells) and expressed as percentage of control condition. PDGF-BB (50 ng/ml) induced PASMC proliferation similar to PDGF-BB (10 ng/ml), and imatinib significantly inhibited the PASMC proliferation induced by PDGF-BB 10 ng/ml or 50 ng/ml. *P = 0.001 versus control; **P < 0.001 versus control; #P = 0.001 versus condition without ST571; ##P < 0.001 versus condition without ST571. NS = not significant.
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Effect of Imatinib on Cultured PASMC Apoptosis
Although H2O2 induced a marked apoptosis (89 ± 7.2% active caspase 3 and 7–containing apoptotic cells), no significant increase in apoptosis was detected in imatinib-treated cells as compared with untreated cells (12.2 ± 5.1% and 10.8 ± 3.8%, respectively; P = 0.68) (Figure 9). Moreover, the measured imatinib-linked inhibition of PASMC proliferation was not due to PASMC cell death, because in our experimental conditions, imatinib did not induce significant caspase 3– and 7–dependent PASMC apoptosis (Figure 9).
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Figure 9. Effects of imatinib (STI571) on pulmonary artery smooth muscle cell (PASMC) apoptosis. The PASMC apoptosis was quantified by the measurement of caspase 3 and 7 activity, detected within whole living cells using ICT's Magic Red substrate-based MR-caspase assay kit. Active caspase 3 and 7–containing cells were red and showed less intense blue nuclei (Hoechst staining) than nonapoptotic cells bearing bright blue nuclei. Results of PASMC apoptosis were expressed as a ratio of active caspase 3 and 7–containing apoptotic PASMCs and total number of PASMCs (% of apoptotic cells). Microphotographs are representative of PASMC apoptosis in control condition (A), imatinib (B), and H2O2 (C) showing active caspase 3 and 7–containing PASMCs. H2O2 induced a marked PASMC apoptosis (positive control) and no difference in PASMC apoptosis was observed in PASMCs treated or not treated with imatinib. *P < 0.0001 versus H2O2. NS = not significant.
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Effect of PDGF and Imatinib on PASMC Migration
PDGF-BB increased PASMC migration (P < 0.001 compared with control) and PDGF-BB–induced PASMC migration was inhibited by imatinib (5 µM), as demonstrated by transwell assay (P < 0.0001; Figure 10). Again, imatinib showed PDGF-specific inhibition because EGF- and bFGF-induced PASMC migration (50 ng/ml) was not affected by imatinib (Figure 10).
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Figure 10. Effect of imatinib on platelet-derived growth factor (PDGF)-B–, EGF-, and bFGF-induced pulmonary artery smooth muscle cell (PASMC) migration (transwell assay). PDGF-BB increased PASMC migration (P < 0.001 compared with control) and PDGF-BB–induced PASMC migration was inhibited by imatinib (5 µM) (P < 0.0001). Imatinib showed PDGF-specific inhibition since EGF- and bFGF- induced PASMC migration (50 ng/ml) were not affected by imatinib. *P < 0.05 versus control; **P < 0.01 versus control; ***P < 0.001 versus control; #P < 0.0001 versus condition without imatinib.
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DISCUSSION
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Our study indicates that PDGF and PDGF receptor mRNA is overexpressed in the pulmonary arteries of human pulmonary hypertensive lungs and that immunostaining localizes PDGF/PDGFR in PASMCs and endothelial cells from pulmonary arteries of patients displaying severe PAH. Phosphorylated (activated) PDGFR proteins are localized into proliferating pulmonary arteries with PCNA and PDGF-B–positive cells. In vitro, PDGF-BB–induced proliferation and migration of cultured human PASMCs are specifically inhibited by imatinib, through blockade of PDGFR phosphorylation. These in vitro effects are not due to the induction of PASMC apoptosis. Because PASMC proliferation and migration are believed to be a major contributor to pulmonary vascular remodeling (neointima formation and media hypertrophy), these findings plead in favor of the potential relevance of PDGF inhibition in the treatment of human PAH.
Many experimental data support the concept that PDGF pathways could play an important role in the pulmonary vascular remodeling process responsible for the progression of PAH (6, 10). Indeed, PDGF is known to induce proliferation of SMCs of different origins (18, 19). Nevertheless, its effects in the proliferation and migration of SMCs are better described in the systemic circulation where it is regarded as an important contributor to major vascular conditions such as atherosclerosis (20). Our present study focused on PASMCs and human idiopathic PAH, to better analyze the role of PDGF in humans, as well as the possible interest of novel therapeutic agents targeting the PDGF pathway, such as imatinib in PAH. Proliferation and migration of PASMCs represent a singular step in the pathogenesis of pulmonary vascular remodeling. Many studies have addressed the phenotypes of the cells involved in neointima formation. Early findings suggested that endothelial cells were the predominant phenotype in plexiform lesions (21), but more recently the role of PASMCs and PASMC migration in neointima formation has been better clarified (22, 23). Our present findings of overexpression of PDGF and PDGF receptors in the pulmonary arterial wall of patients with PAH, together with the demonstration of PDGF pathway activation in PAH vascular lesions (detection of PDGFR-β phosphorylated by immunohistochemistry) associated with cellular proliferation (PCNA-positive cells) and with the confirmation of in vitro PDGF-induced migration and proliferation of PASMCs, support the hypothesis that PDGF is a major contributor of pulmonary vascular remodeling in PAH.
Inhibition of PDGF-induced PASMC migration and proliferation with imatinib supports the possible therapeutic role of PDGF inhibition as a novel approach in PAH, as previously suggested by pioneer studies in monocrotaline-induced pulmonary hypertension in rats (6), as well as by case reports in subjects displaying refractory PAH (12, 13), or severe PAH in the context of chronic myeloid leukemia (14). Imatinib is a competitive inhibitor of the ATP-binding site of PDGF receptor tyrosine kinases that is currently used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors (24, 25). Extracellular signal-related kinase (ERK) phosphorylation has been shown to be a key downstream signal for PDGFR stimulation. It leads to proliferation, inhibition of apoptosis and matrix metalloproteinase activation, a key step for vascular cell migration. Schermuly and colleagues (6) have previously shown that ERK1/2 was strongly suppressed by treatment with 50 mg/kg/day imatinib in rats exposed to monocrotaline, and that imatinib reversed pulmonary hypertension in this experimental model. Because PASMC proliferation and migration are a major characteristic of PAH pathology (2, 3), the effects of imatinib on PDGF-induced PASMC proliferation and migration are presumably relevant to PAH therapy (26).
Currently available PAH therapies have in vitro antiremodeling effects in addition to their vasodilator characteristics (1). However, robust demonstration of antiremodeling effects on pulmonary vascular processes in human PAH is still lacking. Indeed, lung pathology of patients with long-standing treatments with currently approved PAH therapy (including prostaclin derivatives, endothelin receptor antagonists, and type 5 phosphodiesterase inhibitors) still shows major pulmonary vascular remodeling. Because pathology is only available from patients refractory to PAH treatments (i.e., data obtained from lung explant or postmortem specimens), one may claim that patients with a good response to PAH-specific therapy may have better antiremodeling effects. Nevertheless, it is well demonstrated that pulmonary hemodynamics remain extremely abnormal even in patients with long-term beneficial effects of PAH treatment, indicating that pulmonary vascular remodeling is presumably still significant even in good responders (1). To reduce pulmonary vascular remodeling in PAH, growth factor inhibition has to be properly evaluated. This proof-of-concept study in human cells and tissue supports the idea that PDGF-targeted therapies should be tested in future well-designed clinical trials evaluating safety and efficacy in human PAH (26).
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FOOTNOTES
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Supported in part by grants from Ministère de l'Enseignement Supérieur et de la Recherche (GIS-HTAP), Chancellerie des Universités, Legs Poix, and Université Paris-Sud. This research project received financial support from the European Commission under the 6th Framework Program (contract no. LSHM-CT- 2005-018725, PULMOTENSION). This publication reflects only the authors' views and the European Community is in no way liable for any use that may be made of the information contained therein. F.P. is supported by a grant from Ministère de l'Enseignement Supérieur et de la Recherche. R.S. is supported by a grant from European Respiratory Society. The authors thank Steve Pascoe, M.D., M.Sc., Novartis, Horsham, UK, for the kind gift of imatinib.
Originally Published in Press as DOI: 10.1164/rccm.200707-1037OC on April 17, 2008
Conflict of Interest Statement: F.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.D.-G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.L.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. O.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.H. has received  3,000 from Novartis in 2004, 2005, and 2006 to contribute to severe asthma advisory boards. He received 3,000 in 2004, 2005, and 2006 from Novartis to lecture on severe asthma.
Received in original form July 14, 2007;
accepted in final form April 15, 2008
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REFERENCES
|
|---|
- Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004;351:1425–1436.[Free Full Text]
- Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, Reid LM, Tuder RM. Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 2004;43(12, Suppl S):25S–32S.[Abstract/Free Full Text]
- Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, Christman BW, Weir EK, Eickelberg O, Voelkel NF, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43(12, Suppl S):13S–24S.[Abstract/Free Full Text]
- Machado RD, Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips JA III, Newman J, Williams D, Galie N, et al. BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension. Am J Hum Genet 2001;68:92–102.[CrossRef][Medline]
- Humbert M, Monti G, Fartoukh M, Magnan A, Brenot F, Rain B, Capron F, Galanaud P, Duroux P, Simonneau G, et al. Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients. Eur Respir J 1998;11:554–559.[Abstract]
- 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.[CrossRef][Medline]
- Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999;79:1283–1316.[Abstract/Free Full Text]
- Reigstad LJ, Varhaug JE, Lillehaug JR. Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family. FEBS J 2005;272:5723–5741.[CrossRef][Medline]
- Berk BC, Alexander RW, Brock TA, Gimbrone MA Jr, Webb RC. Vasoconstriction: a new activity for platelet-derived growth factor. Science 1986;232:87–90.[Abstract/Free Full Text]
- 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.[Abstract/Free Full Text]
- Druker BJ. Imatinib as a paradigm of targeted therapies. Adv Cancer Res 2004;91:1–30.[CrossRef][Medline]
- Ghofrani HA, Seeger W, Grimminger F. Imatinib for the treatment of pulmonary arterial hypertension. N Engl J Med 2005;353:1412–1413.[Free Full Text]
- Patterson KC, Weissmann A, Ahmadi T, Farber HW. Imatinib mesylate in the treatment of refractory idiopathic pulmonary arterial hypertension. Ann Intern Med 2006;145:152–153.[Free Full Text]
- Souza R, Sitbon O, Parent F, Simonneau G, Humbert M. Long term imatinib treatment in pulmonary arterial hypertension. Thorax 2006;61:736.[Free Full Text]
- Dorfmuller P, Zarka V, Durand-Gasselin I, Monti G, Balabanian K, Garcia G, Capron F, Coulomb-Lhermine A, Marfaing-Koka A, Simonneau G, et al. Chemokine RANTES in severe pulmonary arterial hypertension. Am J Respir Crit Care Med 2002;165:534–539.[Abstract/Free Full Text]
- Eddahibi S, Humbert M, Fadel E, Raffestin B, Darmon M, Capron F, Simonneau G, Dartevelle P, Hamon M, Adnot S. Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 2001;108:1141–1150.[CrossRef][Medline]
- Deshpande NN, Sorescu D, Seshiah P, Ushio-Fukai M, Akers M, Yin Q, Griendling KK. Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle. Antioxid Redox Signal 2002;4:845–854.[CrossRef][Medline]
- Yahiaoui L, Villeneuve A, Valderrama-Carvajal H, Burke F, Fixman ED. Endothelin-1 regulates proliferative responses, both alone and synergistically with PDGF, in rat tracheal smooth muscle cells. Cell Physiol Biochem 2006;17:37–46.[CrossRef][Medline]
- Deng DX, Spin JM, Tsalenko A, Vailaya A, Ben-Dor A, Yakhini Z, Tsao P, Bruhn L, Quertermous T. Molecular signatures determining coronary artery and saphenous vein smooth muscle cell phenotypes: distinct responses to stimuli. Arterioscler Thromb Vasc Biol 2006;26:1058–1065.[Abstract/Free Full Text]
- Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev 2004;15:237–254.[CrossRef][Medline]
- Tuder RM, Groves B, Badesch DB, Voelkel NF. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am J Pathol 1994;144:275–285.[Abstract]
- Yi ES, Kim H, Ahn H, Strother J, Morris T, Masliah E, Hansen LA, Park K, Friedman PJ. Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension: a morphometric and immunohistochemical study. Am J Respir Crit Care Med 2000;162:1577–1586.[Abstract/Free Full Text]
- Nishimura T, Vaszar LT, Faul JL, Zhao G, Berry GJ, Shi L, Qiu D, Benson G, Pearl RG, Kao PN. Simvastatin rescues rats from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle cells. Circulation 2003;108:1640–1645.[Abstract/Free Full Text]
- Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031–1037.[Abstract/Free Full Text]
- Peng B, Lloyd P, Schran H. Clinical pharmacokinetics of imatinib. Clin Pharmacokinet 2005;44:879–894.[CrossRef][Medline]
- Humbert M. Update in pulmonary arterial hypertension 2007. Am J Respir Crit Care Med 2008;177:574–579.[Free Full Text]
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