Platelet-derived Growth Factor Expression and Functionin Idiopathic Pulmonary Arterial Hypertension

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

1Universite 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;2INSERM U764, Clamart, France; 3UPRES EA 2705, Laboratoire de Chirurgie Experimentale, Centre Chirurgical Marie Lannelongue, Universite

Paris-Sud 11, Le Plessis Robinson, France; and 4INSERM U841 and Departement de Physiologie Explorations Fonctionnelles, HopitalHenri-Mondor, Assistance Publique Hopitaux de Paris, Creteil, France

Rationale: Platelet-derived growth factor (PDGF) promotes the pro-liferation and migration of pulmonary artery smooth muscle cells(PASMCs), and may play a role in the progression of pulmonaryarterial hypertension (PAH), a condition characterized by prolifera-tion of PASMCs resulting in the obstruction of small pulmonaryarteries.Objectives: To analyze the expression and pathogenic role of PDGFin idiopathic PAH.Methods: PDGF and PDGF receptor mRNA expression was studied byreal-time reverse transcription–polymerase chain reaction performedon laser capture microdissected pulmonary arteries from patientsundergoing lung transplantation for idiopathic PAH. Immunohisto-chemistry was used to localize PDGF, PDGF receptors, and phos-phorylated PDGFR-b. The effects of imatinib on PDGF-B–inducedproliferation and chemotaxis were tested on human PASMCs.Measurements and Main Results: PDGF-A, PDGF-B, PDGFR-a, andPDGFR-b mRNA expression was increased in small pulmonaryarteries from patients displaying idiopathic PAH, as compared withcontrol subjects.Westernblotanalysis revealedasignificant increasein protein expression of PDGFR-b in PAH lungs, as compared withcontrol lungs. In small remodeled pulmonary arteries, PDGF-A andPDGF-B mainly localized to PASMCs and endothelial cells (perivas-cular inflammatory infiltrates, when present, showed intensivestaining), PDGFR-a and PDGFR-b mainly stained PASMCs and toa lesser extent endothelial cells. Proliferating pulmonary vascularlesions stained phosphorylated PDGFR-b. PDGF-BB–induced pro-liferation and migration of PASMCs were inhibited by imatinib. Thiseffect was not due to PASMC apoptosis.Conclusions: PDGF may play an important role in human PAH. Noveltherapeutic strategies targeting the PDGF pathway should be testedin clinical trials.

Keywords: imatinib; pulmonary arterial hypertension; platelet-derived

growth factor; remodeling; smooth muscle cells

Pulmonary arterial hypertension (PAH) is characterized bya progressive increase in pulmonary vascular resistance leadingto right ventricular failure and ultimately death (1). Remodelingof small pulmonary arteries represents the main pathologicfinding related to PAH with marked proliferation of pulmonaryartery smooth muscle cells (PASMCs), resulting in the obstruc-tion of resistance pulmonary arteries (2, 3). Several mechanismshave been described in the pathogenesis of PAH, includingthose related to current therapeutic targets such as endothelin-1, prostacyclin, and nitric oxide (1, 3). In recent years, recog-nition of germline mutations of genes coding for receptormembers of the transforming growth factor-b superfamily (4)in heritable PAH has emphasized the potential role of growthfactors in the development of PAH. Among them, platelet-derived growth factor (PDGF) has been identified as a novelpossible therapeutic target in PAH (5, 6).

Active PDGF is built up by polypeptides (A and B chain)that form homo- or heterodimers and stimulate a- and b-cellsurface receptors (7). Recently, two additional PDGF geneshave been identified, encoding PDGF-C and PDGF-D poly-peptides (8). PDGF is synthesized by many different cell types,including smooth muscle cells (SMCs), endothelial cells, andmacrophages (7). PDGF has the ability to induce the pro-liferation and migration of SMCs and fibroblasts, and it hasbeen proposed as a key mediator in the progression of severalfibroproliferative disorders such as atherosclerosis, lung fibrosis,and pulmonary hypertension (6–8). It can also induce thecontraction of rat aorta strips in vitro (9).

Novel therapeutic agents such as imatinib mesylate (Gleevec;Novartis, Horsham, UK) inhibit several tyrosine kinases associ-ated with disease states, including BCR-ABL (breakpoint cluster

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

Platelet-derived growth factor (PDGF) plays an importantpart in the progression of experimental pulmonary hyper-tension, but its role in human pulmonary arterial hyper-tension is only partly understood.

What This Study Adds to the Field

Expression of PDGF and PDGF receptors is increased inthe pulmonary arteries of patients with pulmonary arterialhypertension. PDGF induces proliferation and migrationof human pulmonary artery smooth muscle cells, which isinhibited by imatinib, a PDGF receptor inhibitor.

(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

region-Abelson) in patients with chronic myelogenous leukemia,c-kit in patients with gastrointestinal stromal tumors, and PDGFreceptors a and b in patients with certain myeloproliferativedisorders and dermatofibrosarcoma protuberans, respectively(10, 11). Imatinib has been demonstrated to reverse pulmonaryvascular remodeling in animal models of pulmonary hypertension(6) and a few cases of clinical and hemodynamic improvementshave also been reported in human PAH (12–14). Due to the lackof comprehensive human data, we performed a complete analysisof the pathogenic role of PDGF in human PAH. We firstconfirmed increased expression of PDGF and PDGF receptorsby means of real-time reverse transcription–polymerase chainreaction (RT-PCR) performed on laser capture microdissectedpulmonary arteries from lung transplanted patients displayingsevere idiopathic PAH. Western blot analysis showed increasedPDGFR-b protein expression in PAH lungs, as compared withcontrols. Immunohistochemistry techniques were then used tolocalize PDGF-A and PDGF-B and PDGFR-a and PDGFR-bproteins in PAH lungs. The effect of imatinib on PDGFRphosphorylation in cultured PASMCs was studied by Western

blot, and PASMC apoptosis was analyzed by fluorometric de-tection of caspase 3 and 7 activity. Last, we analyzed the effects ofimatinib on PDGF-B–induced proliferation and chemotaxis oncontrol and PAH PASMCs. Our data support the concept thatPDGF is overproduced within the pulmonary artery wall ofpatients 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 5 13) or from tissue obtained during lobectomy or pneumo-nectomy for localized lung cancer (controls, n 5 8), then snap frozen orparaffin embedded, as previously described (15). PASMCs were culturedfrom the same explants as previously described (16). In brief, arteries(diameter: 5–10 mm) were kept in Dulbecco’s modified Eagle medium(DMEM) at 48C before their intimal cell layer and residual adventitialtissue were stripped off using forceps. The dissected media of the vesselswas then cut into small pieces (3–5 mm), which were transferred into cellculture flasks. To allow the PASMCs to grow out, the vessel tissues were

Figure 1. Platelet-derived growth factor (PDGF) and

PDGF receptor (PDGFR) expression in microdissected

pulmonary arteries from patients with severe pulmonaryarterial hypertension (PAH) and from control subjects.

82 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 178 2008

incubated in DMEM supplemented with 20% fetal calf serum (FCS),2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 0.1 mg/mlstreptomycin). After 2 weeks of incubation, the PASMCs collected in theculture medium and the vessel tissues were transferred into new cellculture flasks. This study was approved by the local ethics committee andpatients agreed to contribute to the study.

Laser Capture Microdissection of Pulmonary Arteries, cDNA

Preparation, and RT-PCR

Small pulmonary arteries (100–200 mm) were captured using theASLMD laser microdissection microscope (Leica, Rueil-Malmaison,France). RNA was extracted from microdissected pulmonary arterieswith a PicoPure RNA isolation kit (Arcturus, Mountain View, CA)and then eluted from silicate columns and reverse-transcribed usingSensiscript Reverse Transcription kit (Qiagen, Courtaboeuf, France).Constitutively expressed b-actin was selected as an internal house-keeping gene control for the comparative cycle threshold (CT) method

for the relative quantification of PDGF-A and PDGF-B, and PDGFreceptor a and b. PDGF-A and PDGF-B, PDGF receptor a and b, andb-actin expressions were quantified by RT-PCR with TaqMan geneexpression assays (assay ID number in brackets) (b-actin[Hs99999903_m1], PDGF-A [Hs00234994_m1], PDGFR-a [Hs00183486_m1],PDGF-B [Hs00234042_m1], PDGFR-b [Hs00182163_m1]), and Taq-Man Universal PCR Master Mix performed on an ABI Prism 7000Sequence Detection System (Applied Biosystems, Courtaboeuf,France).

Western Blot

Lung tissue samples from 10 control subjects and 10 patients with PAHwere homogenized in lysis buffer containing 50 mM Tris-HCl, pH 7.4,50 mM NaCl, 1.5 mM ethylenediaminetetraacetic acid, 1% TritonX-100, 3% glycerol, 0.2 mM orthovanadate, and protease inhibitorcocktail (aprotine, leupeptine, and PefaBloc [Roche, Meylan, France]).Lysates were normalized and separated on 8% polyacrylamide gels andtransferred to nitrocellulose membranes. After blocking, the mem-branes were probed with rabbit anti–PDGFR-b (1mg/ml, cat. no. sc432)or anti–phospho-PDGFR-b (1mg/ml, cat. no. sc12909-R) and anti–actin-b loading control (0.2 mg/ml, cat. no. sc-1615) (all from SantaCruz Biotechnology, Inc., Santa Cruz, CA), followed by incubationwith secondary antibodies conjugated with horseradish peroxidase.Bound antibodies were detected by chemiluminescence with the use ofan enhanced chemiluminescence (ECL) detection system (Millipore,Paris, France) and quantified by densitometry.

Immunohistochemistry

Immunohistochemistry was either performed on 8-mm-thick sections offrozen tissue (PDGF-AA and PDGF-BB, and PDGFR-a and PDGFR-b), or on 3-mm-thick sections of paraffin embedded tissue (PDGF-BB,phosphorylated PDGFR-b, proliferating cell nuclear antigen [PCNA]).After routine preparation and microwave unmasking, slides wereprocessed with rabbit anti–PDGF-BB (1 mg/ml, ab15499; Abcam, Paris,France), PDGFR-b (2 mg/ml, sc-432), PDGF-AA (2 mg/ml, sc-128),PDGFR-a (2 mg/ml, sc-338), phospho-PDGFR-b (2 mg/ml, sc-12909-R),and PCNA (2 mg/ml, sc-7907) (all sc catalog numbers are from SantaCruz Biotechnology, Inc.). According to the manufacturer’s recommen-dations, the Envision kit (K4065; Dako, Trappes, France) was used for

Figure 2. Platelet-derived

growth factor receptor(PDGFR)-b protein ex-

pression in total lung of

control subjects and

patients with pulmonaryarterial hypertension

(PAH).

Figure 3. Immunohistochemis-

try: localization of platelet-derivedgrowth factor (PDGF)-A, PDGF-B

and their receptors in pulmonary

arterial lesions of patients with

pulmonary arterial hypertension.(A) Constrictive lesion of a pulmo-

nary artery with perivascular cells

expressing PDGF-A (arrows). (B)Constrictive lesion of a pulmonary

artery: PDGFR-a expression is

mainly detected within the mus-

cular medial layer displaying hy-pertrophy. (C) Plexiform lesion

displaying PDGF-B endothelial cell

expression (arrow). (D) Same le-

sion stained with the anti–PDGFR-b. Note positive smooth muscle

cells within the preserved medial

layer (arrow) and the remodeled

intima (asterisk).

Perros, Montani, Dorfmuller, et al.: PDGF in Pulmonary Arterial Hypertension 83

primary antibody detection. Controls used for these antibodies includedomission of the primary antibody and substitution of the primaryantibody by rabbit IgG.

PASMC Proliferation Assay

PASMCs were serum-starved for 48 hours (0.2% FCS), then incubatedwith PDGF-BB (10 and 50 ng/ml), epithelial growth factor (EGF) orbasic fibroblast growth factor (bFGF) (50 ng/ml; R&D SystemsEurope, Lille, France), with or without 5 mM imatinib (STI571;Novartis, Basel, Switzerland) for 24 hours with [3H]thymidine (Amer-sham France, Les Ulis, France), and cell proliferation was detected bythymidine incorporation (16).

For cell counting, PASMCs were allowed to adhere overnight onLabtek 8 chamber slides (Nunc, Wiesbaden, Germany), then weretreated with the above conditions during 48 days. The chambers werethen removed, the slides washed in phosphate-buffered saline, fixed inacetone, and stained with 49-6-diamidino-2-phenylindole (DAPI). TheDAPI-stained cells were visualized under a Nikon eclipse 80i fluores-cent 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 and7 activity in PASMCs. Caspase activity was detected within whole

living cells using Immunochemistry Technologies (ICT)’s Magic Redsubstrate-based MR-caspase assay kit according to the manufacturer’sinstructions (Serotec, Dusseldorf, Germany). Hydrogen peroxide 1mM was chosen as a positive control for SMC apoptosis (17).

PASMC Migration Assay

PASMC migration was evaluated by the transwell assay. TrypsinizedPASMCs were transferred into the upper chambers of 8-mm-poretranswell plates (VWR, Fontenay-sous-Bois, France). PDGF-BB (10 or100 ng/ml), EGF, or bFGF (50 ng/ml; R&D Systems Europe, Lille,France), with or without 5 mM imatinib, was added to the lowerchamber. After 24 hours at 378C, migration was quantified by countingcells in the bottom of the membrane stained with DiffQuick (DadeBehring S.A., Paris la Defense, France). The number of cells on thelower surface of filter was counted by light microscopy under high-power field (3200). Eight fields were counted in each of three differentexperiments.

Statistical Evaluation

Quantitative variables were presented as means 6 SD. Betweengroups, comparisons were made with Student’s t test; multiple groupcomparisons were performed with analysis of variance and the leastsignificant difference method as a post hoc analysis. P values less than0.05 were considered to reflect statistical significance.

Figure 4. Platelet-derived growth

factor (PDGF)-B and phosphorylatedreceptor expression in pulmonary

arterial lesions of patients with pul-

monary arterial hypertension. (A)

Plexiform lesion displaying PDGF-Bendothelial cell expression within

characteristic endothelium-lined chan-

nels (arrows). (B) Same lesion high-

lighting the proliferating cells throughPCNA (proliferating cell nuclear anti-

gen) staining. (C) A larger pulmonary

artery with medial hypertrophy ex-presses the phophorylated form of

PDGFR-b within smooth muscle cells

(bold arrow) and endothelial cells (thin

arrows). (D) Plexiform lesion with mul-tiple intraluminal endothelium-lined

channels expressing the phophory-

lated form of PDGFR-b within endo-

thelial cells.

Figure 5. Platelet-derived growth

factor (PDGF)-B expression in pul-

monary arterial lesions of patientswith pulmonary arterial hyper-

tension. (A) Small plexiform le-

sion of a pulmonary artery. Notethe perivascular lymphoid infil-

trate (arrows). Hematoxylin–eosin–

saffron staining. (B) Same lesion

showing PDGF-B expression withinlumen-occluding smooth muscle

and endothelial cells. Inflamma-

tory cells to the upper right show

strong staining.

84 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 178 2008

RESULTS

PDGF and PDGF-Receptor mRNA Expression in

Microdissected Pulmonary Arteries

PDGF-A, PDGF-B, PDGFR-a, and PDGFR-b mRNA expres-sion was increased in microdissected small pulmonary arteriesfrom patients displaying severe PAH, as compared with controlsubjects (P , 0.01 for PDGF-B, PDGFR-a, PDGFR-b, andP 5 0.09 for PDGF-A; Figure 1).

PDGF-Receptor Protein Expression in Total Lung

Western blot analysis revealed a significant increase in proteinexpression of PDGFR-b in PAH lungs compared with controllungs. PDGFR-b protein expression normalized to b-actin was0.89 6 0.43 in control lungs and 1.64 6 0.77 in PAH lungs (P 5

0.01) (Figure 2).

Immunohistochemistry

In small pulmonary arteries with constrictive or plexiformremodeling, PDGF-A and PDGF-B expression was mainlylocalized to smooth muscle and endothelial cells (Figures 3A,3C, and 4A). In addition, perivascular inflammatory infiltrates,when present, showed intensive staining (Figures 3A, 5A, and5B). PDGFR-a and PDGFR-b were mainly expressed in SMCsand to a lesser extent in endothelial cells (Figures 3B and 3D).The phosphorylated form of PDGFR-b was detected in bothsmooth muscle and endothelial cells, depending on the pre-dominant proliferating vascular compartment of constrictiveand plexiform lesions (Figures 4C–4D). Proliferating cells were

identified with PCNA and corresponded to endothelial andSMCs within pulmonary vascular lesions (Figure 4B).

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

PDGF-BB 10 ng/ml significantly increased phosphorylatedPDGFR-b protein expression compared with control conditions(P , 0.05). Imatinib given at the same time as PDGF-BBpartially decreased PASMC PDGFR-b phosphorylation in-duced by 10 minutes of PDGF-BB stimulation (P 5 0.06compared with PDGF-BB 10 ng/ml; Figure 6). Imatinib given90 minutes before adding PDGF-BB completely blockedPDGFR-b phosphorylation (P , 0.01 compared with PDGF-BB 10 ng/ml; Figure 6).

Effect of Imatinib on Cultured PASMC Proliferation

PASMC proliferation, induced by PDGF-BB 10 ng/ml, wasinhibited by imatinib (5 mM), as demonstrated by [3H]thymi-dine incorporation assay. To determine the selectivity of STI571on PDGF-induced PASMC proliferation, other growth factors(EGF or bFGF, both at 50 ng/ml) were used to stimulatePASMCs. Although imatinib demonstrated a significant in-hibition of PDGF-BB–induced proliferation (P , 0.0001),proliferation stimulated by EGF or bFGF was unaffected(Figure 7).

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 toPDGF-BB (10 ng/ml) (155.6 6 24.9% and 173.7 6 15.3% ofcontrol conditions, P 5 0.21). Imatinib inhibited significantly

Figure 6. Effect of platelet-derived growth factor (PDGF)-

BB and imatinib (STI571) on PDGFR-b phosphorylation.

Western blot analysis was used to assess expression ofphosphorylated PDGFR-b in pulmonary artery smooth

muscle cells treated with PDGF-BB and imatinib. Phos-

phorylated PDGFR-b protein expression was normalized tob-actin. Immunoblots are representative of phosphory-

lated PDGFR-b expression. PDGF-BB increased phosphor-

ylated PDGFR-b protein expression compared with control

conditions. Imatinib given at the same time as PDGF-BBpartially decreased PDGFR-b phosphorylation induced by

10 minutes of PDGF-BB stimulation, whereas imatinib given

90 minutes before adding PDGF-BB (imatinib pretreatment)

completely blocked PDGFR-b phosphorylation. *P , 0.05versus control; #P , 0.01 versus pretreatment with imatinib.

Perros, Montani, Dorfmuller, et al.: PDGF in Pulmonary Arterial Hypertension 85

the proliferation induced by PDGF-BB 10 ng/ml or 50 ng/ml(P 5 0.001 and P , 0.001, respectively).

Effect of Imatinib on Cultured PASMC Apoptosis

Although H2O2 induced a marked apoptosis (89 6 7.2% activecaspase 3 and 7–containing apoptotic cells), no significantincrease in apoptosis was detected in imatinib-treated cells ascompared with untreated cells (12.2 6 5.1% and 10.8 6 3.8%,respectively; P 5 0.68) (Figure 9). Moreover, the measuredimatinib-linked inhibition of PASMC proliferation was not dueto PASMC cell death, because in our experimental conditions,imatinib did not induce significant caspase 3– and 7–dependentPASMC apoptosis (Figure 9).

Effect of PDGF and Imatinib on PASMC Migration

PDGF-BB increased PASMC migration (P , 0.001 comparedwith control) and PDGF-BB–induced PASMC migration wasinhibited by imatinib (5 mM), as demonstrated by transwellassay (P , 0.0001; Figure 10). Again, imatinib showed PDGF-specific inhibition because EGF- and bFGF-induced PASMCmigration (50 ng/ml) was not affected by imatinib (Figure 10).

DISCUSSION

Our study indicates that PDGF and PDGF receptor mRNA isoverexpressed in the pulmonary arteries of human pulmonaryhypertensive lungs and that immunostaining localizes PDGF/PDGFR in PASMCs and endothelial cells from pulmonaryarteries of patients displaying severe PAH. Phosphorylated(activated) PDGFR proteins are localized into proliferatingpulmonary arteries with PCNA and PDGF-B–positive cells. Invitro, PDGF-BB–induced proliferation and migration of cul-tured human PASMCs are specifically inhibited by imatinib,through blockade of PDGFR phosphorylation. These in vitroeffects are not due to the induction of PASMC apoptosis.Because PASMC proliferation and migration are believed to bea major contributor to pulmonary vascular remodeling (neo-intima formation and media hypertrophy), these findings pleadin favor of the potential relevance of PDGF inhibition in thetreatment of human PAH.

Figure 7. Effect of imatinib on platelet-derived growth

factor (PDGF)-B–, epithelial growth factor (EGF)-, andbasic fibroblast growth factor (bFGF)-induced pulmonary

artery smooth muscle cell (PASMC) proliferation (tritiated

thymidine incorporation assay). Although imatinib dem-

onstrated a significant inhibition of PDGF-BB–inducedproliferation (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.

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 49-6-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 5 0.001 versus control; **P ,

0.001 versus control; #P 5 0.001 versus condition without ST571; ##P ,

0.001 versus condition without ST571. NS 5 not significant.

86 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 178 2008

Many experimental data support the concept that PDGFpathways could play an important role in the pulmonary vascu-lar remodeling process responsible for the progression of PAH(6, 10). Indeed, PDGF is known to induce proliferation of SMCsof different origins (18, 19). Nevertheless, its effects in the pro-

liferation and migration of SMCs are better described in thesystemic circulation where it is regarded as an important con-tributor to major vascular conditions such as atherosclerosis(20). Our present study focused on PASMCs and humanidiopathic PAH, to better analyze the role of PDGF in humans,as well as the possible interest of novel therapeutic agentstargeting the PDGF pathway, such as imatinib in PAH. Pro-liferation and migration of PASMCs represent a singular stepin the pathogenesis of pulmonary vascular remodeling. Manystudies have addressed the phenotypes of the cells involved inneointima formation. Early findings suggested that endothelialcells were the predominant phenotype in plexiform lesions (21),but more recently the role of PASMCs and PASMC migrationin neointima formation has been better clarified (22, 23). Ourpresent findings of overexpression of PDGF and PDGF recep-tors in the pulmonary arterial wall of patients with PAH, to-gether with the demonstration of PDGF pathway activation inPAH vascular lesions (detection of PDGFR-b phosphorylatedby immunohistochemistry) associated with cellular proliferation(PCNA-positive cells) and with the confirmation of in vitroPDGF-induced migration and proliferation of PASMCs, sup-port the hypothesis that PDGF is a major contributor ofpulmonary vascular remodeling in PAH.

Inhibition of PDGF-induced PASMC migration and prolifer-ation with imatinib supports the possible therapeutic role ofPDGF inhibition as a novel approach in PAH, as previouslysuggested by pioneer studies in monocrotaline-induced pulmo-nary hypertension in rats (6), as well as by case reports in subjectsdisplaying refractory PAH (12, 13), or severe PAH in the contextof chronic myeloid leukemia (14). Imatinib is a competitive in-hibitor of the ATP-binding site of PDGF receptor tyrosine ki-nases that is currently used for the treatment of chronic myeloidleukemia and gastrointestinal stromal tumors (24, 25). Extracel-lular signal-related kinase (ERK) phosphorylation has beenshown to be a key downstream signal for PDGFR stimulation.It leads to proliferation, inhibition of apoptosis and matrixmetalloproteinase activation, a key step for vascular cell migra-

Figure 9. Effects of imatinib (STI571) on

pulmonary artery smooth muscle cell

(PASMC) apoptosis. The PASMC apopto-sis was quantified by the measurement of

caspase 3 and 7 activity, detected within

whole living cells using ICT’s Magic Redsubstrate-based MR-caspase assay kit.

Active caspase 3 and 7–containing cells

were red and showed less intense blue

nuclei (Hoechst staining) than nonapop-totic cells bearing bright blue nuclei.

Results of PASMC apoptosis were expressed

as a ratio of active caspase 3 and 7–

containing apoptotic PASMCs and totalnumber of PASMCs (% of apoptotic

cells). Microphotographs are representa-

tive of PASMC apoptosis in control con-dition (A), imatinib (B), and H2O2 (C)

showing active caspase 3 and 7–containing

PASMCs. H2O2 induced a marked PASMC

apoptosis (positive control) and no dif-ference in PASMC apoptosis was ob-

served in PASMCs treated or not treated

with imatinib. *P , 0.0001 versus H2O2.

NS 5 not significant.

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–inducedPASMC migration was inhibited by imatinib (5 mM) (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 versuscontrol; #P , 0.0001 versus condition without imatinib.

Perros, Montani, Dorfmuller, et al.: PDGF in Pulmonary Arterial Hypertension 87

tion. Schermuly and colleagues (6) have previously shown thatERK1/2 was strongly suppressed by treatment with 50 mg/kg/dayimatinib in rats exposed to monocrotaline, and that imatinibreversed pulmonary hypertension in this experimental model.Because PASMC proliferation and migration are a major char-acteristic of PAH pathology (2, 3), the effects of imatinib onPDGF-induced PASMC proliferation and migration are pre-sumably relevant to PAH therapy (26).

Currently available PAH therapies have in vitro antiremod-eling effects in addition to their vasodilator characteristics (1).However, robust demonstration of antiremodeling effects onpulmonary vascular processes in human PAH is still lacking.Indeed, lung pathology of patients with long-standing treat-ments with currently approved PAH therapy (including pros-taclin derivatives, endothelin receptor antagonists, and type 5phosphodiesterase inhibitors) still shows major pulmonary vascu-lar remodeling. Because pathology is only available from patientsrefractory to PAH treatments (i.e., data obtained from lungexplant or postmortem specimens), one may claim that patientswith a good response to PAH-specific therapy may have betterantiremodeling effects. Nevertheless, it is well demonstrated thatpulmonary hemodynamics remain extremely abnormal even inpatients with long-term beneficial effects of PAH treatment,indicating that pulmonary vascular remodeling is presumably stillsignificant even in good responders (1). To reduce pulmonaryvascular remodeling in PAH, growth factor inhibition has to beproperly evaluated. This proof-of-concept study in human cellsand tissue supports the idea that PDGF-targeted therapies shouldbe tested in future well-designed clinical trials evaluating safetyand efficacy in human PAH (26).

Conflict of Interest Statement: F.P. does not have a financial relationship witha 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 aninterest in the subject of this manuscript. P.D. does not have a financialrelationship with a commercial entity that has an interest in the subject of thismanuscript. I.D.-G. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. C.T. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. J.L.P. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. M.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. E.F. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. S.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. O.M. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. P.H. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. D.E. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. S.E. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. G.S. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. R.S. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. M.H. has received V3,000 from Novartis in 2004, 2005, and2006 to contribute to severe asthma advisory boards. He received V3,000 in2004, 2005, and 2006 from Novartis to lecture on severe asthma.

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