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Platelet-Derived Growth Factor Expression and Function in Idiopathic Pulmonary Arterial Hypertension

July 2, 2008

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-beta. 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-alpha, and PDGFR-beta mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects.Western blot analysis revealeda significant increase in protein expression of PDGFR-beta 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-alpha and PDGFR-beta mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-beta. 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.

Keywords: imatinib; pulmonary arterial hypertension; platelet- derived growth factor; remodeling; smooth muscle cells

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- b superfamily (4) in heritable PAH has emphasized the potential role of growth factors in the development of PAH. Among them, plateletderived 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 alpha- and beta-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 alpha and beta 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-beta protein expression in PAH lungs, as compared with controls. Immunohistochemistry techniques were then used to localize PDGF-A and PDGF-B and PDGFR-alpha and PDGFR-beta 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.

METHODS

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[degrees]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 [mu]m) 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 beta-actin was selected as an internal housekeeping gene control for the comparative cycle threshold (CTau) method for the relative quantification of PDGF-A and PDGF-B, and PDGF receptor alpha and beta. PDGF-A and PDGF-B, PDGF receptor alpha and beta, and beta-actin expressions were quantified by RT-PCR with TaqMan gene expression assays (assay ID number in brackets) (beta- actin [Hs99999903_m1], PDGF-A [Hs00234994_m1], PDGFR-alpha [Hs00183486_m1], PDGF-B [Hs00234042_m1], PDGFR-beta [Hs00182163_m1]), and Taq-Man 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-beta (1[mu]g/ml, cat. no. sc432) or anti-phospho-PDGFR-beta (1[mu]g/ml, cat. no. sc12909-R) and anti-actin-beta loading control (0.2 [mu]g/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-mm-thick sections of frozen tissue (PDGF-AA and PDGF-BB, and PDGFR-alpha and PDGFRb), or on 3-[mu]m-thick sections of paraffin embedded tissue (PDGF-BB, phosphorylated PDGFR-beta, proliferating cell nuclear antigen [PCNA]). After routine preparation and microwave unmasking, slides were processed with rabbit anti-PDGF-BB (1 [mu]g/ml, ab15499; Abcam, Paris, France), PDGFR-beta (2 [mu]g/ml, sc-432), PDGF-AA (2 [mu]g/ ml, sc-128), PDGFR-alpha (2 [mu]g/ml, sc-338), phospho-PDGFR-beta (2 [mu]g/ml, sc-12909-R), and PCNA (2 [mu]g/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 [mu]M 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-[mu]m-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 [mu]M imatinib, was added to the lower chamber. After 24 hours at 37[degrees]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 highpower field ( x 200). 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.

RESULTS

PDGF and PDGF-Receptor mRNA Expression in Microdissected Pulmonary Arteries

PDGF-A, PDGF-B, PDGFR-alpha, and PDGFR-beta mRNA expression was increased in microdissected small pulmonary arteries from patients displaying severe PAH, as compared with control subjects (P

PDGF-Receptor Protein Expression in Total Lung

Western blot analysis revealed a significant increase in protein expression of PDGFR-beta in PAH lungs compared with control lungs. PDGFR-beta protein expression normalized to beta-actin was 0.89 +- 0.43 in control lungs and 1.64 +- 0.77 in PAH lungs (P = 0.01) (Figure 2).

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-alpha and PDGFR-beta were mainly expressed in SMCs and to a lesser extent in endothelial cells (Figures 3B and 3D). The phosphorylated form of PDGFR-beta 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).

Effect of PDGF-BB and Imatinib on PDGFR-beta Phosphorylation

PDGF-BB 10 ng/ml significantly increased phosphorylated PDGFR- beta protein expression compared with control conditions (P

Effect of Imatinib on Cultured PASMC Proliferation

PASMC proliferation, induced by PDGF-BB 10 ng/ml, was inhibited by imatinib (5 [mu]M), 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

We confirmed the inhibitory effect of imatinib on PDGFBB-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

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).

Effect of PDGF and Imatinib on PASMC Migration

PDGF-BB increased PASMC migration (P

DISCUSSION

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-beta 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).

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 V3,000 from Novartis in 2004, 2005, and 2006 to contribute to severe asthma advisory boards. He received V3,000 in 2004, 2005, and 2006 from Novartis to lecture on severe asthma.

AT A GLANCE COMMENTARY

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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26. Humbert M. Update in pulmonary arterial hypertension 2007. Am J Respir Crit Care Med 2008;177:574-579. Frederic Perros1,2,3, David Montani1,2, Peter Dorfmuller1,2, Ingrid Durand-Gasselin2, Colas Tcherakian1,2, Jerome Le Pavec1, Michel Mazmanian3, Elie Fadel3, Sacha Mussot3, Olaf Mercier3, Philippe Herve3, Dominique Emilie2, Saadia Eddahibi4, Gerald Simonneau1, Rogerio Souza1,2, and Marc Humbert1,2

1 Universite Paris-Sud 11, UPRES EA 2705, Centre National de Reference de l’Hypertension Arterielle Pulmonaire, Service de Pneumologie et Reanimation Respiratoire, Institut Paris-Sud Cytokines, Hopital Antoine-Beclere, Assistance Publique Hopitaux de Paris, Clamart, France; 2 INSERM U764, Clamart, France; 3 UPRES EA 2705, Laboratoire de Chirurgie Experimentale, Centre Chirurgical Marie Lannelongue, Universite Paris-Sud 11, Le Plessis Robinson, France; and 4 INSERM U841 and Departement de Physiologie Explorations Fonctionnelles, Hopital Henri-Mondor, Assistance Publique Hopitaux de Paris, Creteil, France

(Received in original form July 14, 2007; accepted in final form April 15, 2008)

Supported in part by grants from Ministere de l’Enseignement Superieur et de la Recherche (GIS-HTAP), Chancellerie des Universites, Legs Poix, and Universite 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 Ministere de l’Enseignement Superieur 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.

Correspondence and requests for reprints should be addressed to Marc Humbert, M.D., Ph.D., Service de Pneumologie et Reanimation Respiratoire, Hopital Antoine-Beclere, 157 rue de la Porte de Trivaux, 92140 Clamart, France. E-mail: marc.humbert@abc.aphp.fr

Am J Respir Crit Care Med Vol 178. pp 81-88, 2008

Originally Published in Press as DOI: 10.1164/rccm.200707-1037OC on April 17, 2008

Internet address: www.atsjournals.org

Copyright American Thoracic Society Jul 1, 2008

Originally published by Perros, Frederic Montani, David; Dorfmuller, Peter; Durand- Gasselin, Ingrid; Tcherakian, Colas; Pavec, Jerome Le; Mazmanian, Michel; Fadel, Elie; Mussot, Sacha; Mercier, Olaf; Herve, Philippe; Emilie, Dominique; Eddahibi, Saadia; Simonneau, Gerald; Souza, Rogerio.

(c) 2008 American Journal of Respiratory and Critical Care Medicine. Provided by ProQuest Information and Learning. All rights Reserved.




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