Study Population

We studied lung specimens obtained during lung transplantation in 8 patients with iPH (4 women, 4 men) and during lobectomy or pneumonectomy for localized lung cancer in 9 control subjects (2 women, 7 men). In the lung specimens from control subjects, pulmonary arteries were studied at a distance from tumor areas. Age (mean±SD) was 46±12 years in the patients with iPH and 53±10 years in the control subjects. The mean pulmonary artery pressure in the group with iPH was 63±11 mm Hg (range, 48 to 85 mm Hg), mean pulmonary vascular resistance was 24.5±3 mm Hg · L⁻¹ · min⁻¹ · m⁻² (range, 16 to 37 mm Hg · L⁻¹ · min⁻¹ · m⁻²), and mean cardiac index was 2.0±0.2 L · min⁻¹ · m⁻² (range, 1.6 to 3 L · min⁻¹ · m⁻²). Transthoracic echocardiography was performed preoperatively in the control subjects to rule out PH. None of the patients with iPH had been treated with appetite suppressants, and 6 were treated with PGI2 at the time of transplantation. Patients with a mutation in the bone morphogenic protein receptor II (BMPRII) gene were excluded from this study. The study was approved by our institutional review board.

Isolation and Culture of Human P-ECs

Human P-ECs were obtained by Dispase I digestion (Roche Diagnostics, Penzbeg, Germany) 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 MCDB131 medium (Invitrogen, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS), 50 U/mL of penicillin/streptomycin, 4 mmol/L l-glutamine, 25 mmol/L HEPES, 10 U/mL heparin, 1 μg/mL human endothelial cell growth supplement, and 10 ng/mL vascular endothelial growth factor (Promocell, Heidelberg, Germany). Immunomagnetic purification of P-ECs was then performed with anti-PECAM-1 (CD31) monoclonal antibody–labeled DynaBeads¹⁴ (Dynal, Biotech, Compiègne, France). To characterize the endothelial cell phenotype, cells were labeled with acetylated low-density lipoprotein (LDL) coupled to a fluorescent carbocyanine dye (DiI-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, Lyon, France).¹⁴ Experiments also were performed using monoclonal antibodies against desmin and vimentin (Dako, Trappes, France). Cells positive for DiI-Ac-LDL and UEA-1 and negative for desmin and vimentin were labeled as endothelial cells and constituted >95% of our culture cells. Cells were used for the study between passages 3 and 6.

Culture of PA-SMCs

PA-SMCs were cultured from explants as previously described.⁶ To determine the phenotypic characteristics of cultured PA-SMCs, the cells from each culture were assessed for expression of muscle-specific contractile and cytoskeletal proteins, including smooth muscle cell a-actin, desmin, and vinculin.⁶

Measurements of PA-SMC Proliferation

PA-SMCs in Dulbecco’s modified Eagle’s medium supplemented with 15% FCS were seeded in 24-well plates at a density of 5×10⁴ cells per well and allowed to adhere. The cells were subjected to 48 hours of growth arrest in medium containing 0% FCS and then treated with 1 mL conditioned medium from P-ECs with or without fluoxetine (10⁻⁵ mol/L). 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 5×10⁴ cells per well and allowed to adhere and grow in the same medium as described above (but without heparin) for 24 hours. The P-ECs were then serum starved in MCDB131 medium with or without the tryptophan hydroxylase (TPH) inhibitor p-chlorophenylalanine (PCPA, 10 mmol/L) for the next 24 hours. PA-SMC proliferation was assessed with or without 10⁻⁵ mol/L fluoxetine and with or without Bosentan (10⁻⁵ mol/L). PA-SMC proliferation was also assessed in response to 5% FCS with or without fluoxetine (10⁻⁵ mol/L) or PCPA (10⁻⁵ mol/L) added 20 minutes before FCS. The doses of fluoxetine (10⁻⁵ mol/L) and PCPA (10⁻⁵ mol/L) were chosen according to previous studies showing selective inhibition of 5-HTT^6,12 and TPH^15–17 activities, respectively. For each condition, [³H]thymidine 0.6 μCi/mL was added to each well. After incubation for 24 hours, the cells were washed twice with phosphate-buffered saline (PBS), treated with ice-cold 10% trichloroacetic acid, and dissolved in 0.1 N NaOH (0.5 mL/well). The incorporated radioactivity was counted. [³H]thymidine incorporation into DNA is reported as counts per minute per well.

Determination of TPH Activity

P-ECs in MCDB131 medium supplemented as described above were seeded in 6-well plates at a density of 300 000 cells/mL per well for 24 hours. The cells were then subjected to growth arrest in MCDB131 medium (0% FCS) with the decarboxylase inhibitor NSD1015 (3-hydroxybenzylhydrazine dihydrochloride, 50 μmol/L), NSD1015+L-tryptophan (10 μmol/L), or NSD1015 (50 μmol/L)+L-tryptophan (10 μmol/L)+PCPA (10 μmol/L) for 2 hours at 37°C. Cells were scraped in their own medium (1 mL), 50 μL of high-performance liquid chromatography (HPLC) solution (2 mol/L perchloric acid, 0.1% EDTA, 0.1% Na₂S₂0₅, wt/vol) was added, and the mix was frozen immediately at −80°C. The reaction product, 5-hydroxy-tryptophan (5-HTP), in P-EC homogenates was measured with HPLC coupled with electrochemical detection (HPLC-ECD) as previously described.¹⁸

Measurements of 5-HT and ET-1 Concentrations in P-EC Medium

The P-EC medium used to measure 5-HT and ET-1 levels was obtained from serum-starved cells in MCDB131 medium (0% FCS) for 24 hours with or without PCPA (10 to 50 μmol/L). ET-1 levels in P-EC media were determined by ELISA (R&D Systems, Lille, France), and 5-HT levels were measured by HPLC-ECD. Absolute values were calculated from 5-HT peak areas with reference to authentic serotonin standards and were not corrected for recovery.¹⁸

Total RNA Isolation

Total RNA was isolated from lung, P-ECs, and PA-SMCs with the Trizol reagent (Invitrogen, Cergy-Pontoise, France) and estimated from optical density measurements (260- to 280-nm absorbance ratio). To avoid inappropriate amplification of residual genomic DNA, total RNA was systemically treated by DNase. Total RNA was extracted from P-ECs and PA-SMCs with the Qiagen RNeasy Mini kit (QIAGEN SA, Courtaboeuf, France) according to the manufacturer’s instructions. RNA concentration was determined by standard spectrophotometric techniques, and RNA integrity was assessed by visual inspection of ethidium bromide–stained denaturing agarose gels. RNAs from human intestine, liver, and cerebral tissues were purchased from Clontech, BD-Biosciences (Mountain View, Calif).

cDNA Preparation and Real-Time Quantitative Polymerase Chain Reaction

First-strand cDNA synthesis was carried out with the SuperScript II Reverse Transcriptase System (Life Technologies, Inc, Gaithersburg, Md). A mixture containing 1 μg total RNA, 2 μL deoxynucleotide triphosphate mix (10 mmol/L), and 100 ng random primers in a total volume of 12 μL was incubated for 5 minutes at 65°C and chilled on ice. Then, 4 μL of 1st Strand Buffer, 2 μL of DTT (0.1 mol/L), and 40 U of ribonuclease inhibitor (RNAse-Out, Invitrogen, Carlsbad, Calif) were added to the samples, which were then heated at 42°C for 2 minutes. After 1 μL SuperScript reverse transcriptase II (200 U/μL) was added, the mixture was incubated for 10 minutes at 25°C, 50 minutes at 42°C, and 15 minutes at 70°C. The cDNA was diluted 1:20 for use in real-time quantitative polymerase chain reaction (PCR). Amplification was performed in triplicate with an ABI Prism 7000 (Applied Biosystems, Foster City, Calif). Predeveloped sequence detection reagents specific for human tph1, tph2, and 5-HTT (Assays-on-Demand Gene Expression Products, Applied Biosystems), including forward and reverse primers, and a TaqMan MGB probe (fluorophores dye-labeled) were purchased as mixtures and used at 1.25 μL/25 μL PCR. Each 25 μL PCR reaction mix also included 1× TaqMan universal PCR master mix (P/N4304437, Applied Biosystems). The thermal cycle conditions were 2 minutes at 50°C for optimal AmpErase UNG activity, 10 minutes at 95°C to activate the AmpliTaq polymerase, and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The fluorescent signal at each cycle generated by the release of fluorophores from the quencher caused by the 5′-exonuclease activity of AmpliTaq polymerase was plotted against the cycle number. The threshold cycle Ct, defined as the cycle number at which an increase above background fluorescence could be reliably detected, was determined for each sample with GeneAmp software. Relative quantification was performed with the comparative ΔΔCt method by normalization with 18s ribosomal RNA.

Lung Immunohistochemical Labeling of TPH and 5-HTT

Paraffin sections (5 mm thick) of lung specimens were mounted on Superfrost Plus slides (Fisher Scientific, France). For 5-HTT immunostaining, the slides were dewaxed in 100% toluene, and the sections were then rehydrated by immersion in decreasing ethanol concentrations (100%, 95%, and 70%) and then in distilled water. Endogenous peroxidase activity was blocked with H₂O₂ in methanol (0.3% vol/vol) for 10 minutes. After 3 washes with PBS, the sections were preincubated in PBS supplemented with 3% (wt/vol) bovine serum albumin for 30 minutes and then incubated overnight at 4°C with polyclonal rabbit anti-TPH or goat anti–5-HTT antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif) diluted 1:1000 in PBS containing 0.02% bovine serum albumin. The sections were exposed for 1 hour to biotin-labeled anti-goat secondary antibodies (Dako, France) diluted 1:200 in the same buffer and then to streptavidin biotin horseradish peroxidase solution. Peroxidase staining was carried out with 3,3′-diaminobenzidine tetrahydrochloride dihydrate (DAB, Sigma, St Louis, Mo) and hydrogen peroxide. Finally, the sections were stained with hematoxylin and eosin.

Statistical Analyses

All data are reported as mean±SEM. To assess the effects of treatment with the medium derived from P-ECs of patients and control subjects on the proliferation of PA-SMCs from patients and control subjects, ANOVA was used for between-group comparisons. When ANOVA indicated significance and an interaction, the groups were compared by use of a nonparametric test. To assess the effects of various treatments on smooth muscle cell growth induced by EC media, 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. The nonparametric Mann-Whitney test was used to compare patients and control subjects in the other parts of the study. Values of P<0.05 were considered statistically significant.

The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written

Growth of PA-SMCs in Response to Serum-Free Media Derived From Cultured P-ECs

Serum-free media from cultured P-ECs obtained from the lungs of patients with iPH and control subjects were added to PA-SMCs cultured in 0% serum. This produced a marked increase in [³H]thymidine incorporation (Figure 1A). The increase was larger when PA-SMCs were stimulated with media of P-ECs from patients with iPH compared with P-ECs from control subjects. Moreover, PA-SMCs from patients with iPH grew faster than those from control subjects in response to serum-free medium from homologous P-ECs. The highest growth rate was observed for PA-SMCs from patients with iPH treated with serum-free medium from homologous P-ECs (Figure 1A). To determine the contribution of ET-1 to P-EC media-stimulated PA-SMC growth, we first tested the endothelin receptor antagonist Bosentan (Figure 1B), which did not alter the total growth response. Treatment with Bosentan also did not affect PA-SMC growth (data not shown). To evaluate the contribution of 5-HT, we added fluoxetine (10⁻⁵ mol/L) to inhibit 5-HTT in PA-SMCs. This diminished the growth response to P-EC medium by ≈60%. With fluoxetine, growth rates of PA-SMCs from control subjects were identical with media of P-ECs from control subjects and from patients with iPH (Figure 1C). Fluoxetine also reduced the growth-stimulating action of serum, which contains serotonin (Figure 1D). Levels of both ET-1 and 5-HT were markedly increased in the media of P-ECs from patients with iPH compared with those from control subjects (Figure 2A).

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Figure 1. Growth of PA-SMCs from patients with iPH and control subjects (C) in response to serum-free media derived from cultured P-ECs from patients with iPH or from control subjects. A, [³H]thymidine incorporation into PA-SMCs from control subjects (open bars) and from patients with iPH (hatched bars) after exposure to conditioned media from cultured EC-iPH or control subjects (EC-C). *P<0.01, **P<0.001 vs basal conditions in nonstimulated PA-SMCs; ‡P<0.01 vs values for PA-SMCs of either origin stimulated with EC-C. B, [³H]thymidine incorporation into PA-SMCs from control subjects (open bars) and from patients with iPH (hatched bars) after exposure to medium from EC-Cs, medium from EC-Cs with Bosentan (Bos) (10⁻⁵ mol/L), medium from EC-iPHs, and medium from EC-iPH Cs with Bosentan (10⁻⁵ mol/L). There were no differences between values obtained with and without Bosentan. C, [³H]thymidine incorporation into PA-SMCs from control subjects (open bars) and from patients with iPH (hatched bars) after exposure to medium from EC-Cs, medium from EC-Cs incubated with 10⁻⁵ M PCPA (EC-C+PCPA), medium from EC-Cs with 10⁻⁵ mol/L fluoxetine (EC-C+Fluox), medium from EC-iPH, medium from EC-iPH incubated with p-chlorophenylalanine (EC-iPH+PCPA), and medium from EC-iPH with fluoxetine (EC-iPH+Fluox). ‡P<0.01 vs corresponding values without PCPA or fluoxetine. D, [³H]Thymidine incorporation into PA-SMCs from patients with iPH (hatched bars) and from control subjects (open bars) after exposure to 5% FCS. The response also was measured in the presence of fluoxetine (10⁻⁵ mol/L) or PCPA (10⁻⁵ mol/L). *P<0.05 vs values for cells stimulated by 5% FCS. All values are mean±SEM of 5 patients and 5 control subjects. Each experiment involved testing serum-free media from cultured P-ECs from an individual on PA-SMCs from the same individual.

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Figure 2. Synthesis of 5-HT and ET-1 in P-ECs from patients with iPH and control subjects. A, 5-HT and ET-1 levels measured in conditioned media derived from cultured P-ECs from patients with iPH (n=8) or control subjects (n=9) after a 24-hour incubation. Each bar is mean±SEM. *P<0.05, **P<0.01 vs control subjects. B, TPH activity in P-ECs from patients with iPH and control subjects. Experiments were performed in the presence of the decarboxylase inhibitor NSD1015 (50 μmol/L). *P<0.01 vs control subjects. C, Pathway for serotonin biosynthesis, indicating the target enzymes PCPA and NSD1015.

Evidence for Serotonin Synthesis by P-ECs

To investigate whether 5-HT measured in P-EC serum-free media was released from an indoleamine pool stored in P-ECs or was synthesized de novo by these cells, experiments were performed after P-EC treatment with PCPA,¹⁵ a selective inhibitor of TPH, the rate-limiting enzyme of 5-HT biosynthesis. P-EC pretreatment with PCPA (10⁻⁵ mol/L) reduced 5-HT to undetectable levels in the medium and reduced PA-SMC growth to an extent similar to that obtained by PA-SMC pretreatment with fluoxetine (Figure 1C). No additive inhibitory effect occurred when the 2 drugs were combined. We also tested possible effects of fluoxetine and PCPA on the growth-stimulating action of serum, which contains nanomolar concentrations of serotonin (Figure 1D). The difference in serum-induced PA-SMC growth between patients with iPH and control subjects was abolished by fluoxetine (as previously reported⁶) but was not affected by PCPA, demonstrating that PCPA did not induce nonspecific effects on PA-SMC growth.

We then measured TPH activity in P-EC homogenates by quantifying 5-HTP accumulation after addition of NSD1015, an inhibitor of aromatic L-amino acid decarboxylase¹⁹ (Figure 2B and 2C). Only after the substrate L-tryptophan was added to the incubation mixture did 5-HTP become detectable. Accumulation of 5-HTP was >3 times greater in P-ECs from patients with iPH than from control subjects. Addition of 10 μmol/L of the TPH inhibitor PCPA nearly abolished 5-HTP accumulation in P-ECs from control subjects but induced only 50% inhibition in P-ECs from patients with iPH. Neither 5-HTP accumulation nor 5-HT release was detected using PA-SMCs from patients with iPH or control subjects.

Expression of TPH in Lungs and Cultured Cells From Patients With iPH

TPH is encoded by 2 genes, the well-characterized tph1 gene expressed in enterochromaffin cells²⁰ and the recently identified tph2 gene expressed in the central nervous system.²¹ Using real-time quantitative PCR to measure tph1 and tph2 mRNA, we found that tph2 mRNA was detected selectively in cerebral tissues, whereas tph1 mRNA was detected predominantly in the gut and, to a lesser extent, in whole-lung homogenates and cerebral tissues from humans. No significant amounts of tph1 mRNA were detected in the liver (Figure 3A). Levels of tph1 mRNAs in whole-lung homogenates from patients with iPH were increased ≈3-fold compared with control subjects (Figure 3B). Expression of tph1 in quiescent cultured P-ECs also was markedly increased in patients with iPH compared with control subjects (Figure 3B). Staining with toluidine blue was negative, ruling out contamination of P-EC cultures by mast cells. No tph1 mRNA was detected in PA-SMCs from patients with iPH or control subjects. In contrast, 5-HTT expression predominated in PA-SMCs and was greater in PA-SMCs from patients with iPH than from control subjects (Figure 3B).

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Figure 3. Expression of TPH and 5-HTT in lungs and cultured cells from patients with iPH and control subjects. A, Relative levels of tph1 and tph2 mRNAs in human lung, liver, intestine, or cerebral tissue. B, Relative levels of 5-HTT and tph1 mRNAs in lung tissues, cultured P-ECs, and cultured PA-SMCs from patients with iPH (n=8) and control subjects (n=9). Values are mean±SEM of arbitrary units. *P<0.05, **P<0.01 vs control subjects.

Immunolocalization of TPH and 5-HTT in Lungs From Patients With iPH

Immunohistochemical analyses revealed that TPH immunostaining was confined to the intima of pulmonary vessels; no staining was seen in the media (Figure 4). Even in remodeled vessels with marked muscular hypertrophy from iPH patients, tph1-like immunoreactivity was present in the intima, whereas it was less marked in obstructed vessels in which endothelium was absent or replaced by a neointima. In contrast, 5-HTT immunostaining was located predominantly in the media in lung specimens from patients with iPH and from control subjects, producing uniform staining in the media of vessels with marked muscular hypertrophy (Figure 4).

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Figure 4. Immunolocalization of TPH and 5-HTT in lung sections from control subjects and patients with iPH. In a lung section from a control subject (A) and in lung sections from patients with iPH (B through D), TPH immunostaining was confined to pulmonary endothelial cells and was absent in obstructed vessels lacking an endothelium (D). E, A lung section from a control subject with 5-HTT–like immunoreactivity located mainly in smooth muscle cells. F through H, In lung sections from patients with iPH, 5-HTT–like immunoreactivity is still prominent in the medial layer of pulmonary arteries with marked muscular hypertrophy. Scale bar=100 μm in all sections.