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Angiopoietin/Tie2 Pathway Influences Smooth Muscle Hyperplasia in Idiopathic Pulmonary Hypertension

Institut National de la Santé et de la Recherche Médical U651 and Département de Physiologie, Hôpital H. Mondor, Assistance Publique-Hôpitaux de Paris, Créteil; UPRES-EA 2705, Service de Chirurgie Thoracique, Vasculaire, et de Transplantation Cardiopulmonaire, Hôpital Marie Lannelongue, Le Plessis Robinson; Service de Pneumologie, Hôpital A. Béclère, Assistance Publique-Hôpitaux de Paris, Clamart; and UMR 677, Institut National de la Santé et de la Recherche Médical/Université Pierre et Marie Curie, NeuroPsychoPharmacologie, Faculté de Médecine Pitié-Salpêtrière, Paris, France; and Laboratory of Physiology, Faculty of Medicine, Free University of Brussels, Brussels, Belgium

Correspondence and requests for reprints should be addressed to Laurence Dewachter, M.Sc., INSERM U651, Faculté de Médecine, 8, Rue Général Sarrail, 94010 Créteil, France. E-mail: dewachter{at}creteil.inserm.fr

	ABSTRACT

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Rationale: Angiopoietins are involved in blood vessel maturation and remodeling.

Objectives: One consequence of endothelium-specific tyrosine kinase-2 (Tie2) receptor activation by angiopoietin-1 (Ang1) is the release of endothelium-derived growth factors that recruit vascular wall cells. We investigated this process in idiopathic pulmonary arterial hypertension (iPAH).

Methods: Ang1, Ang2, and total and phosphorylated Tie2 expression (mRNA and protein) was evaluated in human lung specimens and in cultured pulmonary artery smooth muscle cells (PA-SMCs) and pulmonary endothelial cells (P-ECs) isolated from patients with iPAH and control subjects. Media collected from Ang1-treated P-ECs were assessed for their PA-SMC growth-promoting effect.

Measurements and Main Results: Tie2 receptor was fourfold higher in lungs and P-ECs from patients with iPAH than in those from control subjects, with a parallel increase in phosphorylated lung Tie2 receptor. In contrast, Ang1 and Ang2 expression in lungs, P-ECs, and PA-SMCs did not differ. Incubation of PA-SMCs with medium collected from P-EC cultures induced marked proliferation, and this effect was stronger when using P-ECs from patients with iPAH than from control subjects. Ang1 pretreatment of P-ECs from either patients or control subjects induced a further increase in PA-SMC proliferation. Fluoxetine, an inhibitor of the mitogenic action of serotonin, reduced the growth-promoting effect of P-EC media. Ang1 added to P-ECs from patients with iPAH increased the production of endothelin-1 (ET-1) and serotonin, but not of platelet-derived growth factor-BB or epidermal growth factor, and increased the amount of mRNA encoding tryptophan hydroxylase-1 (the rate-limiting enzyme of serotonin synthesis), preproET-1, and ET-1–converting enzyme.

Conclusions: The Ang1/Tie2 pathway is potentiated in iPAH, contributing to PA-SMC hyperplasia via increased stimulation of endothelium-derived growth factors synthesis by P-ECs.

Key Words: angiopoietin-1 • growth factors • idiopathic pulmonary arterial hypertension • pulmonary artery smooth muscle • pulmonary endothelial cells

Idiopathic pulmonary arterial hypertension (iPAH) is a rare disease with a grim prognosis and a poorly understood pathogenesis. The disease is characterized by abnormal vascular cell proliferation that eventually obliterates the small pulmonary arteries (1). Although hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is considered the main component of the remodeling process in iPAH, the nature of the primary abnormality that triggers and perpetuates PA-SMC proliferation in iPAH is unclear. Whether smooth muscle hyperplasia results from inherent characteristics of PA-SMCs (2–5) or from dysregulation of molecular events governing PA-SMC growth, such as signals originating from pulmonary endothelial cells (P-ECs), remains an open question (6, 7).

The role of P-ECs in angiogenesis and remodeling is now better elucidated. A critical aspect of vessel development is maturation, during which P-ECs no longer proliferate or migrate but instead promote vessel stabilization by recruiting periendothelial support cells, which differentiate into smooth muscle cells (6). Because this maturation phase is under the control of angiopoietin-1 (Ang1), which acts selectively on ECs via the endothelium-specific tyrosine kinase-2 (Tie2) receptor (8), one theory is that EC stimulation by Ang1 leads to the release of signaling molecules that act on smooth muscle cells (9, 10). In accordance with a role for such a mechanism in the pulmonary circulation, it was shown that P-ECs stimulated with Ang1 were capable of promoting PA-SMC growth (10). In a more recent study, we obtained evidence that serum-free medium of quiescent human P-ECs elicited marked PA-SMC proliferation, although the P-ECs were not previously stimulated by Ang1. Interestingly, this effect was greater with P-ECs from patients with iPAH than with P-ECs from control subjects, suggesting dysregulation of this mechanism in iPAH (11). Thus, an intriguing hypothesis is that the angiopoietin/Tie2 pathway, which seems to play a role in normal vessel wall development, may be altered in iPAH, thereby leading to excessive smooth muscle proliferation.

In the present study, we investigated the angiopoietin-1/Tie2 pathway in patients with iPAH. For this purpose, we sampled lung tissue from patients with iPAH and control subjects and we first investigated the expression of Ang1, Ang2, and Tie2 receptor in cultured PA-SMCs and in P-ECs, as well as in whole lung homogenates. Second, we investigated whether treatment of cultured P-ECs with Ang1 increased the growth-promoting activity of culture medium from P-ECs of patients and control subjects. Third, in studies by Sullivan, Eddahibi, and coworkers, serotonin (5-HT) appeared as the main P-EC–derived growth factor acting on PA-SMCs (10, 11). We therefore investigated whether Ang1 altered the release by P-ECs of 5-HT and other growth factors including endothelin-1 (ET-1), platelet-derived growth factor-BB (PDGF-BB), and epidermal growth factor (EGF). Because 5-HT synthesis is mediated by the enzyme tryptophan hydroxylase-1 (TPH1), and because ET-1 activation and maturation are controlled by endothelin-converting enzyme-1 (ECE-1), we also examined the effect of Ang1 on mRNA levels of these enzymes, as well as on mRNA levels of preproendothelin-1 (preproET-1). We selected patients meeting a strict definition of iPAH, because it is unclear whether the pulmonary vascular biological derangements are identical in iPAH and in associated PAH or secondary PAH.

	METHODS

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Tissue Collection
Lung tissue was sampled at lung transplantation in 14 patients with iPAH (7 females and 7 males) and during lobectomy or pneumonectomy for a localized tumor in 15 control patients (5 females and 10 males). In the control subjects, transthoracic echocardiography was performed preoperatively to rule out pulmonary hypertension, and pulmonary arteries were studied at a distance from tumor areas. Mean pulmonary artery pressure in the group with iPAH was 70 ± 10 mm Hg (range, 50 to 85 mm Hg), mean pulmonary vascular resistance was 34 ± 6 mm Hg · L^–1 · min^–1 · m^–2 (range, 22 to 51 mm Hg · L^–1 · min^–1 · m^–2), and mean cardiac index was 2.0 ± 0.3 L · min^–1 · m^–2 (range, 1.5 to 3.1 L · min^–1 · m^–2). None of the patients with iPAH had been treated with appetite suppressants; 10 patients were receiving PGI2 at the time of transplantation. None of the patients had bone morphogenetic protein receptor type II (BMPRII) or activin-like kinase type 1 (ALK-1) mutations. The study was approved by our institutional review board.

Isolation of PA-SMCs and P-ECs
Human PA-SMCs were cultured from explants of pulmonary arteries as previously described (12). To determine the phenotypic characteristics of cultured PA-SMCs, the cells were assessed for expression of muscle-specific contractile and cytoskeletal proteins including smooth muscle cell -actin, desmin, and vinculin (12). PA-SMCs were used between passages 3 and 6.

Human P-ECs were obtained by Dispase I (Roche Diagnostics, Penzbeg, Germany) digestion of a 5-cm³ lung tissue fragment left at 37°C overnight. The suspension was filtered, plated onto 0.1% gelatin-coated wells, and grown in MCDB 131 medium (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS), penicillin/streptomycin (50 U/ml), 4 mM L-glutamine, 25 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, heparin (10 U/ml), endothelial cell growth supplement (1 µg/ml), and vascular endothelial growth factor (10 ng/ml) (PromoCell, Heidelberg, Germany). Immunomagnetic purification of P-ECs was performed with anti-platelet endothelial cell adhesion molecule-1 (CD31) monoclonal antibody–labeled DynaBeads (Dynal Biotech, Compiegne, France). To characterize the endothelial cell phenotype, P-ECs were labeled with acetylated low-density lipoprotein coupled to a fluorescent carbocyanine dye (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [Dil-Ac– LDL]; Tebu, Le Perray en Yvelines, France) and stained with antibodies against the endothelial cell–specific lectin Ulex europaeus agglutinin-1 (UEA-1; Sigma-Aldrich, Ayrshire, UK) (13). Experiments were also performed with monoclonal antibodies against desmin and vimentin (Dako, Glostrup, Denmark). Cells with positive staining for Dil-Ac–LDL and UEA-1 and negative staining for desmin and vimentin were taken as endothelial cells and constituted more than 95% of our P-EC cultures. Cells were used between passages 3 and 6 (11).

Total RNA Extraction
Total RNA was prepared from snap-frozen human lung tissue samples (weight, 100 mg) by homogenization according to the method of Chomczynski and Sacchi (14), using TRIzol reagent (Invitrogen, Cergy-Pontoise, France). Total RNA was extracted from PA-SMCs and P-ECs, using an RNeasy Mini kit (Qiagen SA, Courtaboeuf, France), according to the manufacturer's instructions. The RNA concentration was determined by standard spectrophotometric techniques and RNA integrity was assessed by visual inspection of ethidium bromide–stained agarose gels.

cDNA Preparation and Quantitative Real-Time Polymerase Chain Reaction
First-strand cDNA synthesis was performed with the SuperScript II RT (reverse transcriptase) system (Invitrogen). A mix containing 1 µg of total RNA, 2 µl of deoxynucleotide triphosphate mix (10 mM), and 100 ng of random primers in a total volume of 12 µl was incubated for 5 min at 65°C and chilled on ice. After addition of 4 µl of first-strand buffer, 2 µl of dithiothreitol (0.1 M), and 40 U of RNase inhibitor (RNaseOUT; Invitrogen), the samples were heated at 42°C for 2 min. Finally, after addition of 1 µl of SuperScript II RT (200 units/µl), the reaction was incubated for 10 min at 25°C, 50 min at 42°C, and 15 min at 70°C. The cDNA was diluted 1:20 for use in quantitative RT-polymerase chain reaction (PCR).

Predeveloped sequence detection reagents specific for human Ang1,Tie2, and TPH1 (Assays-on-Demand gene expression products; Applied Biosystems, Foster City, CA) including forward and reverse primers, as well as a TaqMan MGB probe (FAM dye labeled), were supplied as mixtures and were used at 1.25 µl per 25-µl PCR. Each 25-µl PCR mix also included 1x TaqMan universal PCR master mix (Applied Biosystems). Ang2, PreproET-1, and ECE-1 primers for PCR were designed with Primer Express software (Applied Biosystems). To avoid inappropriate amplification of residual genomic DNA, intron-spanning primers were selected and internal control 18S rRNA primers were provided. For each sample, the amplification reaction was performed in triplicate with SYBR Green mix (Applied Biosystems) and specific primers. Signal detection and analysis of results were performed with ABI PRISM 7000 sequence detection software (Applied Biosystems). Relative quantification was achieved with the comparative C_t method by normalization with 18S ribosomal RNA.

Protein Extraction and Ang1, Ang2, and Tie2 Western Blotting
Proteins were extracted from snap-frozen tissue samples (weight, 100 mg) by homogenization in an appropriate amount of homogenizing buffer (Complete Mini, protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany] in phosphate-buffered saline [PBS] and 0.1% Triton X-100). The homogenates were centrifuged at 4°C and the supernatants were collected. PA-SMC and P-EC homogenates were prepared by the same procedure. After determination of the protein concentration, using the method of Bradford (15), 100 µg of protein from each lung sample was resuspended in 3x Laemmli buffer, boiled for 5 min, and separated on 10% acrylamide gels by electrophoresis. Proteins were electrophoretically transferred to a nitrocellulose membrane (Sigma-Aldrich) for 1 h at room temperature. After blocking with 5% bovine serum albumin in 1x Tween (T)–Tris buffered saline (TBS: 10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) for 2 h at room temperature, the membrane was washed three times with T–TBS at room temperature for 5 min. The membrane was incubated with goat anti-human Ang1, Ang2, and rabbit anti-human Tie2 antibody (diluted 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight with rocking. Quantitative Tie2 phosphorylation immunoblotting was performed with anti-phosphotyrosine antibody (Cell Signaling Technology, Danvers, MA). The membrane was then washed three times for 5 min each and incubated with secondary antibody (rabbit anti-goat or goat anti-rabbit IgG conjugated with horseradish peroxidase, diluted 1:2,000; Dako, Glostrup, Denmark) for 1 h at room temperature. Immunoreactive bands were detected with an enhanced chemiluminescence Western blotting analysis system (GE Healthcare UK, Little Chalfont, UK) and quantified by laser densitometry. Relative quantification was performed by normalization with -actin (Sigma-Aldrich).

Lung Tie2 Receptor Immunolocalization
Lung specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections were dewaxed and progressively rehydrated. For antigen retrieval, sections were incubated in target retrieval buffer (Dako, Glostrup, Denmark) and heated in a bath for 20 min at 97°C. Endogenous peroxidase activity was quenched with hydrogen peroxide in PBS (1%, vol/vol) for 10 min, and the sections were blocked by incubation with bovine serum albumin in PBS (3%, wt/vol) for 30 min. Sections were allowed to react at 37°C for 1 h with rabbit anti-human Tie2 antibody (diluted 1:100 in PBS; Tebu-Bio, Le Perray en Yvelines, France). The sections were then incubated with biotinylated anti-rabbit IgG (Dako, Le Perray en Yvelines, France) and subsequently with streptavidin–peroxidase (Dako). Antibody binding was detected with a liquid DAB (diaminobenzidine) substrate kit (AbCvs Biology, Paris, France). The appearance of a brown reaction product was observed under a light microscope. Nuclei were counterstained with hematoxylin and mounted. Negative controls run without the primary antibody were tested.

PA-SMC Growth Assays
PA-SMCs in Dulbecco's modified Eagle's medium supplemented with 10% FCS were seeded at a density of 5 x 10⁴ cells per well in 24-well plates and allowed to adhere. The cells were subjected to 48 h of growth arrest in medium containing 0% FCS and then treated with 1 ml of conditioned medium collected from P-ECs with or without Ang1 treatment (50 ng/ml). P-EC serum-free medium was obtained as follows: at the time of initiating PA-SMC growth arrest, P-ECs were seeded in 24-well plates at a density of 5 x 10⁴ cells per well and were allowed to adhere and to grow in supplemented MCDB 131 (as described above but without heparin) for 24 h. The P-ECs were then subjected to 24 h of growth arrest in MCDB 131 medium with 0.2% FCS, with or without Ang1 treatment (50 ng/ml). PA-SMC proliferation was then assessed with or without fluoxetine (10^–6 mol/L) and with or without bosentan (10^–6 mol/L). Neither of these drugs was used to treat P-ECs. PA-SMC proliferation was also assessed in response to 5% FCS. The fluoxetine dose (10^–6 mol/L) was chosen on the basis of previous studies showing selective inhibition of 5-HT transporter (5-HTT) with this dose (12, 16). For each condition, [³H]thymidine (0.6 µCi/ml) was added to each well. After incubation for 24 h, the cells were washed twice with PBS, treated with ice-cold 10% trichloroacetic acid, and neutralized with 0.1 N NaOH (0.5 ml/well). [³H]thymidine incorporation into DNA was counted and reported as counts per minute per well. A concentration of 50 ng/ml was chosen in these experiments on the basis of preliminary studies of Ang1 concentrations in relation to the growth-promoting activity of P-EC medium on PA-SMCs.

Effects of Ang1 on 5-HT, ET-1, EGF, and PDGF-BB Synthesis by Cultured P-ECs
P-ECs were seeded as described above and allowed to adhere. The cells were synchronized with medium containing 0.2% FCS for 24 h and then either left untreated or treated with a range of Ang1 concentrations (1–50 ng/ml; human recombinant angiopoietin-1; R&D Systems, Minneapolis, MN). The supernatants were collected and used for quantitation of 5-HT, ET-1, EGF, and PDGF-BB. Serotonin (5-HT) levels were measured by high-performance liquid chromatography coupled to electrochemical detection. Absolute values were calculated on the basis of 5-HT peak areas with reference to authentic 5-HT standards and were not corrected for recovery (17). ET-1, EGF, and PDGF-BB concentrations in P-EC supernatant were determined by ELISA (R&D Systems). For assays of TPH1, preproET-1, and ECE-1 mRNAs, P-ECs were seeded and synchronized, as described above. The cells were exposed to Ang1 (50 ng/ml) for 4 h and then used for quantitative RT-PCR.

Statistical Analysis
All data are reported as means ± SEM. Analysis of variance was used for comparisons between two groups. When the analysis of variance ratio reached p < 0.05, the groups were compared using nonparametric Mann-Whitney U tests. p Values less than 0.05 were considered statistically significant. To assess the effects of various treatments on PA-SMC growth induced by P-EC medium, the nonparametric Kruskal-Wallis test was performed. When the Kruskal-Wallis test showed a significant difference, the groups were further compared using a nonparametric Student-Newman-Keuls test.

	RESULTS

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Ang1, Ang2, and Tie2 Receptor Expression in Human Lung Homogenates and Cultured Cells
Ang1 expression as assessed by quantitative RT-PCR and Western blotting for mRNA and protein, respectively, in whole lung tissue homogenates and on PA-SMCs was not different in patients with iPAH and in control subjects (Figures 1 and 4). Ang1 mRNA was not detected in P-ECs from patients with iPAH or control subjects. Ang2 expression assessed by quantitative RT-PCR and Western blotting was similar in lung tissue homogenates, as well as in P-ECs from patients with iPAH and control subjects (Figures 2 and 4). In contrast, Tie2 receptor expression in whole lung tissue homogenates and P-ECs was increased three- to fourfold in patients with iPAH compared with control subjects (Figures 3 and 4). About 60% of lung Tie2 receptor was phosphorylated. As shown in Figure 3, immunohistochemical analyses revealed that Tie2 immunostaining was confined to the intima of pulmonary vessels, in both patients with iPAH and control subjects; no staining was seen in the media. No relationship was found between hemodynamic parameters (pulmonary artery pressure and pulmonary vascular resistance) and levels of Ang1, Ang2, or Tie2 protein in the lung.

View larger version (20K): [in this window] [in a new window]   Figure 1. Expression of angiopoietin-1 (Ang1) in lungs from patients with idiopathic pulmonary arterial hypertension (iPAH; n = 14; solid columns) and control subjects (CT; n = 15; open columns). Relative Ang1 mRNA (A) and Ang1 protein (B) levels are shown. Values represent means ± SEM, in arbitrary units.

View larger version (20K): [in this window] [in a new window]   Figure 2. Expression of angiopoietin-2 (Ang2) in lungs from patients with iPAH (n = 14; solid columns) and control subjects (n = 15; open columns). Relative Ang2 mRNA (A) and Ang2 protein (B) levels are shown. Values represent means ± SEM, in arbitrary units.

View larger version (30K): [in this window] [in a new window]   Figure 3. Expression and immunolocalization of Tie2 receptor (Tie2) in lungs from patients with iPAH (n = 14; solid columns) and control subjects (n = 15; open columns). (A) Relative levels of Tie2 mRNA; (B) relative levels of Tie2 and phosphorylated Tie2 (P-Tie2) proteins. Values represent means ± SEM, in arbitrary units. *p < 0.05, **p < 0.01 compared with control subjects. (C) Tie2 receptor immunostaining in lung sections from control subjects (CT) and patients with iPAH. Tie2 receptor immunostaining is detected only in the endothelium of pulmonary vessels. Scale bars: 100 µm.

View larger version (19K): [in this window] [in a new window]   Figure 4. Expression of Ang1, Ang2, and Tie2 in cultured pulmonary artery smooth muscle cells (PA-SMCs) or pulmonary microvascular endothelial cells (P-ECs) from patients with iPAH (n = 6; solid columns) and control subjects (n = 7; open columns). (A and D) Relative Ang1 mRNA (A) and Ang1 protein (D) levels in PA-SMCs. No expression of Ang1 was detected in P-ECs. (B and E) Relative Ang2 mRNA (B) and Ang2 protein (E) levels in P-ECs. No expression of Ang2 was detected in PA-SMCs. (C and F) Relative Tie2 mRNA (C) and Tie2 protein (F) levels in P-ECs. Values represent means ± SEM, in arbitrary units. *p < 0.05, compared with control subjects.

View larger version (19K): [in this window] [in a new window]   Figure 5. [3H]thymidine incorporation by PA-SMCs from control subjects in response to serum-free medium (open column) after exposure to conditioned media from cultured P-ECs from control subjects (gray columns) and patients with iPAH (solid columns) maintained for 24 h with Ang1 (50 ng/ml) or without Ang1 (A). The same experiments were performed using cotreatment of P-ECs with bosentan (10–6 mol/L) (B) and fluoxetine (10–6 mol/L) (C). *p < 0.05; **p < 0.01 compared with the basal level (without Ang1 treatment); p < 0.05 compared with values obtained with medium of cultured P-ECs in the absence of Ang1; p < 0.05 compared with values obtained with medium of cultured P-ECs in the presence of Ang1 (50 ng/ml).

View larger version (18K): [in this window] [in a new window]   Figure 6. Synthesis of serotonin (5-HT; A), endothelin-1 (ET-1; B), epidermal growth factor (EGF; C), and platelet-derived growth factor-BB (PDGF-BB; D) in P-ECs from patients with iPAH (n = 5; solid columns) and control subjects (n = 6; open columns) with or without Ang1 (1–100 ng/ml) treatment, after 24 h of incubation. Each column represents the mean ± SEM. *p < 0.05, **p < 0.01 compared with control subjects; p < 0.05, p < 0.01 compared with the basal level (without Ang1 treatment).

View larger version (11K): [in this window] [in a new window]   Figure 7. Expression of mRNAs encoding tryptophan hydroxylase-1 (TPH1; A), preproendothelin-1 (prepro-ET-1; B), and endothelin-converting enzyme-1 (ECE-1; C) in cultured P-ECs isolated from patients with iPAH and control subjects and treated (open columns) or not treated (solid columns) with Ang1 (50 ng/ml). Values represent means ± SEM, in arbitrary units. *p < 0.05, **p < 0.01 compared with control subjects; p < 0.05 compared with the basal level (without Ang1 treatment).

	DISCUSSION

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The present study builds on our previous finding that cultured P-ECs constitutively produce and release growth factors that act on PA-SMCs (11). During development, factors derived from endothelial cells participate in blood vessel formation and maturation by recruiting and stabilizing vascular wall cells (18). Because this maturation phase of angiogenesis is known to be under the control of Ang1, one current hypothesis is that the angiopoietin pathway may be abnormal in PAH (10, 19, 20), leading to excessive smooth muscle proliferation. Evidence for this hypothesis is lacking, however. In previous studies, we found that serum-free medium of quiescent P-ECs elicited marked PA-SMC proliferation, and that this effect was greater with P-ECs from patients with iPAH than from control subjects (11). Because these results were obtained without stimulating P-ECs with Ang1, we reasoned that the excessive release of growth factors seen in P-ECs from patients with iPAH was due to an intrinsic P-EC abnormality independent of Ang1 stimulation. However, in the present study, we found that stimulation of PA-SMC proliferation by culture medium from P-ECs was more marked when P-ECs were pretreated with Ang1, whether the P-ECs were from control subjects or from patients with iPAH. Together with the results obtained by Sullivan and coworkers in rat SMCs (10), these observations strongly support a physiologic role for Ang1 in the pulmonary circulation involving stimulation of P-ECs to release growth factors that act on PA-SMCs. The physiological importance of these observations is also supported by the fact that the proliferation-promoting effect of Ang1 occurred at doses previously shown to induce Tie2 receptor signaling, EC migration, decreased EC permeability, and protection against EC apoptosis (21–24).

The Ang1/Tie2 pathway has been reported to be abnormal in PAH. Ang1 was overexpressed in proportion to pulmonary vascular resistance in patients with various forms of PAH (19). Moreover, increased Ang1 expression was found in piglets with high flow–induced PAH (25). In the present study, no increase was noted in Ang1 mRNA or protein level in lung tissue or PA-SMCs from patients with iPAH, in comparison with control subjects. The reasons for these apparently discrepant results are not clear. An important point is that, in contrast to results reported by Du and coworkers, who failed to detect Ang1 in samples from normal subjects (19), we detected significant amounts of Ang1 in lungs and PA-SMCs from our control subjects. Moreover, in our 14 patients with iPAH, we found no correlation between iPAH severity and Ang1 levels, in contrast to the findings by Du and coworkers. It is also important to point out that Ang1 expression in the present study was assessed on isolated cultured PA-SMCs, in the absence of mechanical stress or pharmacologic influences, and that the PA-SMCs were from patients with strictly defined iPAH. Our results therefore strongly suggest that Ang1 expression may remain unchanged in the majority of patients with advanced iPAH.

In contrast to the absence of changes in lung Ang1 expression, Tie2 receptor expression was increased in lungs and cultured P-ECs from patients with iPAH compared with control subjects. This overexpression of Tie2 was conserved in cultured P-ECs, in the absence of possible confounding influences by in vivo hemodynamic conditions, therapies, or concomitantly expressed mediators. One expected effect of Tie2 activation in P-ECs is increased release of factors that promote the recruitment and proliferation of vascular wall cells. To investigate this possibility, we evaluated the consequences of Tie2 activation by Ang1 on the release of ET-1, 5-HT, PDGF-BB, and EGF by P-ECs. The last two growth factors were chosen because PDGF-BB is known to play a critical role in the recruitment of pericytes to newly formed vessels (26, 27) and because EGF has been shown to undergo up-regulation in response to Ang1 in endothelial cells (28). In the present study, however, neither PDGF-BB nor EGF was affected by stimulating the Tie2 receptor with Ang1, whether the P-ECs were from patients with iPAH or from control subjects. Moreover, basal growth factor release by P-ECs did not differ between patients with iPAH and control subjects. In contrast, and as previously demonstrated, ET-1 and 5-HT concentrations in the medium of quiescent P-ECs from patients with iPAH were markedly higher than in medium from control subjects (11). Stimulation with Ang1 further increased the release of ET-1 and 5-HT from P-ECs of patients with iPAH, whereas in P-ECs from control subjects it stimulated only the release of 5-HT. The 5-hydroxyindoleacetic acid:5-HT ratio remained unchanged, indicating that the 5-HT increase was not due to altered indoleamine degradation. Ang1 also increased the mRNAs encoding preproendothelin-1, ECE-1, and TPH1 in P-ECs from patients with iPAH, but not in those from control subjects. These results are in accordance with those obtained by Sullivan and coworkers, showing that Ang1 stimulated 5-HT release by P-ECs (10). The biphasic effect of Ang1 on ET-1 levels is consistent with previous studies showing increased nitric oxide formation with high Ang1 doses (29). Here, we show that the increased 5-HT release induced by Ang1 occurred together with increased expression of TPH1, the rate-limiting enzyme of 5-HT synthesis. Moreover, Ang1 also stimulated the release of ET-1 as a result of increased expression of ECE1, and to a lesser extent, preproET-1. It is noteworthy that the basal levels of the two mediators stimulated by Ang1 in P-EC media, namely, 5-HT and ET-1, differed between patients with iPAH and control subjects, whereas those of PDGF and EGF, which were not up-regulated by Ang1, were similar in patients and control subjects. A link between these alterations and Tie2 overexpression in P-ECs from patients with iPAH is unlikely, because basal 5-HT and ET-1 levels were measured in the absence of Tie2 activation by Ang1. Of note is the fact that Ang1 is synthesized mostly by smooth muscle cells, whereas Ang2, which works as an antagonist of the Tie2 receptor, is synthesized mostly by endothelial cells. Thus, it is unlikely that endogenous Ang1 contributed in our in vitro system to activate Tie2 receptors. Whatever the case, the present results provide evidence that the Ang1/Tie2 pathway is altered in patients with iPAH in a way that leads to excessive release of potent factors involved in the contraction and remodeling of pulmonary blood vessels, namely, 5-HT and ET-1.

In our previous study investigating the cross-talk between P-ECs and PA-SMCs, we found that 5-HT accounted for 60% of the growth-promoting effect of P-EC medium on PA-SMCs, with no contribution from ET-1 (11). Because the mitogenic action of 5-HT on PA-SMCs is mediated by its selective transporter (5-HTT) (12), we determined whether the increased growth activity of P-EC medium induced by Ang1 was abolished by treating the PA-SMCs with fluoxetine, a selective 5-HTT inhibitor. Fluoxetine dramatically reduced the growth-promoting effect of P-EC medium on PA-SMCs under all study conditions but did not abolish the stimulating activity of Ang1. Moreover, in the presence of fluoxetine, PA-SMC proliferation induced by EC medium from patients with iPAH remained slightly higher than the proliferation induced by EC medium from control subjects in the absence of fluoxetine. These results indicate that Ang1 stimulated growth factors other than 5-HT, which largely contributed to the growth-promoting activity of the P-EC medium. It is therefore likely that pathways other than those mediated by 5-HT are involved in the proliferative response of PA-SMCs to P-EC medium and that Ang1 stimulates several of these pathways, thereby contributing to pulmonary vessel muscularization. It is also likely that EC-SMC cross-talk involving 5-HT is unique to the pulmonary circulation, because TPH expression seems lower in systemic vessel endothelial cells than in pulmonary vessels, and because 5-HT does not seem to exert potent mitogenic effects on SMCs in systemic vessels (30, 31).

Because the present findings were obtained from in vitro studies, they do not readily allow definite conclusions about the role of angiopoietins in the pulmonary hypertension process. In accordance with the proposal that the Ang1/Tie2 pathway contributes to pulmonary vascular remodeling, rats engineered to overexpress Ang1 in the pulmonary circulation developed severe pulmonary hypertension with prominent medial hypertrophy (10, 20). Moreover, rats expressing a soluble inactive form of the Tie2 receptor were protected against monocrotaline-induced PAH (32). In contrast, administration of cells expressing Ang1 to monocrotaline-treated rats was shown to induce protection against the development of pulmonary hypertension (33). One aspect not completely clarified in the present study is that the final effects of Ang1 in vivo also depend on the action of the Tie2 antagonistic ligand Ang2. Indeed, Ang2 has been identified as a functional antagonist of Ang1, and one established concept is that Tie2 receptor activation in vivo results from the delicate balance between Ang1 and Ang2 levels. In the present study, we found that lung Ang2 and Ang1 protein levels did not differ between patients with iPAH and control subjects, suggesting that the balance was not affected by iPAH. However, Ang1 is expressed chiefly by PA-SMCs and acts in a paracrine fashion, whereas Ang2, which is expressed mainly by ECs and stored in Weibel-Palade bodies, acts via an autocrine mechanism. Although higher levels of lung phosphorylated Tie2 in patients with iPAH argue for increased in vivo activation of the receptor, it is difficult to extrapolate our in vitro findings to in vivo effects of angiopoietins in human iPAH. Moreover, Ang1 has many biological functions, including inhibition of endothelial apoptosis, which may protect the pulmonary vasculature under some experimental conditions. Ang1 has been reported to exert antiinflammatory effects (22, 34, 35) in addition to regulatory effects on vessel maturation and vascular quiescence. On the other hand, Ang2 was shown to sensitize endothelial cells to tumor necrosis factor- and to play a crucial role in the induction of inflammation (36). Further studies are therefore needed to better delineate the exact role played by Ang1 in experimental and human PAH.

In the present study of human tissues, we obtained evidence that the Ang1/Tie2 pathway was abnormal in iPAH and contributed to PA-SMC hyperplasia through excessive release of growth factors by P-ECs. Because Ang1 is now considered a pericyte-derived paracrine signal for the endothelium, these findings identify Ang1/Tie2 pathway abnormalities as a component of the dysregulation of cross-talk between endothelial and smooth muscle cells. Further studies are needed to better understand the importance of these abnormalities in the process of aberrant smooth muscle proliferation that characterizes the pathogenesis of PAH.

	FOOTNOTES

 
Supported by grants from INSERM, the Ministère de la Recherche, the Institut des Maladies Rares, the Délégation à la Recherche Clinique de l'AP-HP, and the Belgian Foundation for Cardiac Surgery. This study/research project received financial support from the European Commission under the 6th Framework Programme (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 herein. L.D. was a fellow of the European Respiratory Society.

Originally Published in Press as DOI: 10.1164/rccm.200602-304OC on August 17, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 28, 2006; accepted in final form August 16, 2006

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