Abstract
Rationale: Inflammatory cytokines may affect pulmonary vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH). CC chemokine ligand 2 (CCL2) is synthesized by vascular cells and can stimulate monocyte/macrophage migration and smooth muscle cell (SMC) proliferation.
Objectives: To investigate the role of CCL2 in IPAH.
Methods: CCL2 levels in plasma, monocytes, lungs, and medium from pulmonary endothelial cell (P-EC) or pulmonary artery SMC (PA-SMC) cultures were measured by ELISA and Western blot analysis. CCL2 receptor CCR2 mRNA levels in monocytes, P-ECs, and PA-SMCs were measured by real-time polymerase chain reaction. Effect of CCL2 on PA-SMC proliferation and migration was assessed using [3H]thymidine incorporation and a modified Boyden's chamber. The effect of endothelial cell–derived CCL2 on monocyte migration was measured using a modified Boyden's chamber.
Measurements and Main Results: Compared with control subjects, we found the following in patients with IPAH: elevated CCL2 protein levels in plasma and lung tissue, whereas monocyte CCL2 levels were similar between patients and control subjects, and elevated CCL2 release by P-ECs or PA-SMCs. P-ECs released twice as much CCL2 than did PA-SMCs. Monocyte migration was markedly increased in the presence of P-ECs, and the increase was larger with P-ECs from patients with IPAH. CCL2-blocking antibodies reduced P-ECs' chemotactic activity by 60%. Compared with controls, PA-SMCs from patients exhibited stronger migratory and proliferative responses to CCL2, in keeping with the finding that CCR2 was markedly increased in PA-SMCs from patients.
Conclusions: These results suggest that CCL2 overproduction may be a feature of the abnormal P-EC phenotype in IPAH, contributing to the inflammatory process and to pulmonary vascular remodeling.
Accumulating evidence suggests a role for inflammation in the pathogenesis of pulmonary arterial hypertension. One major cytokine synthesized by vascular cells and able to stimulate monocyte/macrophage migration is CC chemokine ligand 2 (CCL2).
Idiopathic pulmonary arterial hypertension is associated with an overproduction of CCL2. Pulmonary endothelial cells are a major source of CCL2, which behaves as chemoattractant for circulating inflammatory cells and as growth factor for pulmonary artery smooth muscle cells.
The mechanisms underlying pulmonary vessel infiltration by monocytes/macrophages are unclear, and the role for inflammatory cells in pulmonary vascular remodeling remains to be elucidated. Inflammation may occur as a secondary event during progression of PAH or may develop as part of the intrinsic vessel-wall cell abnormalities associated with IPAH. In a recent study, we found that pulmonary ECs (P-ECs) constitutively released growth factors that acted on pulmonary artery SMCs (PA-SMCs) (8). Whether intrinsic P-EC abnormalities during IPAH include the expression and release of inflammatory cytokines remains to be further investigated. One of the major cytokines synthesized by vascular cells and acting as a potent mediator of monocyte/macrophage activation and migration is CC chemokine ligand 2 (CCL2) (formerly called monocyte chemoattractant protein [MCP]-1), which has been implicated in a wide range of chronic inflammatory processes, including atherosclerosis (9). The effects of CCL2 are mediated through the CC chemokine receptor 2 (CCR2), which is expressed by many cell types including monocytes and vascular SMCs (10). Evidence for a critical role of CCL2 in experimental PAH was recently obtained in monocrotaline-exposed rats, in which antibodies or antisense oligonucleotides directed against CCL2 prevented the occurrence of pulmonary vascular remodeling (11). The role for CCL2 in pulmonary vascular remodeling in human IPAH is unknown, and the mechanism leading to increased CCL2 production in patients with IPAH has not been identified.
Here, we measured plasma and lung CCL2 levels in patients with IPAH to determine whether the abnormal pulmonary vascular cell phenotype in IPAH was associated with CCL2 overproduction. The effects of EC-derived CCL2 on migration of monocytes, and on migration and proliferation of PA-SMCs, were assessed in vitro.
This study was approved by our institutional review board (Hôpital Henri Mondor, Creteil, France). All patients and control subjects signed an informed consent document before study inclusion.
A detailed description of the methods is available in the online supplement.
Circulating CCL2 levels were measured in 44 patients (30 women and 14 men with a mean ± SD age of 46 ± 15 yr) with IPAH (mean pulmonary artery pressure (), 52 ± 12 mm Hg [range, 32–91]; mean cardiac index, 1.95 ± 0.5 L/min/m2; and mean pulmonary vascular resistance [PVR], 28.3 ± 9 Wood units/m2) before initiation of specific PAH therapy and in 17 healthy control subjects (10 women and 7 men aged 47 ± 8 yr). Measurements were repeated in a subgroup of nine patients 1 month after initiation of continuous prostacyclin infusion. Patients who had PAH associated with another condition (e.g., connective tissue disease or liver disease) were not included in the study.
Lung specimens were collected during lung transplantation in eight patients with IPAH (5 men and 3 women aged 35 ± 14 yr) and during lobectomy or pneumonectomy for localized lung cancer in eight control subjects (6 men and 2 women aged 56 ± 12 yr). In the group with IPAH, was 57 ± 17 mm Hg (range, 31–83 mm Hg), mean cardiac index was 2.3 ± 0.7 L/min/m2, and mean PVR was 27.2 ± 9.6 Wood units/m2. All eight patients with IPAH received prostacyclin at the time of transplantation; none of them had mutations in the bone morphogenic protein receptor II (BMPRII) gene. PAH was ruled out in the control subjects based on echocardiography findings before surgery. In the lung specimens from control subjects, pulmonary arteries were studied at a distance from the tumor.
Human PA-SMCs were cultured from explants of pulmonary arteries, and P-ECs were isolated using immunomagnetic purification and cultured as previously described (8, 12). Cells were used between passages 3 and 6.
Mononuclear cells were isolated from venous blood of healthy donors and patients with IPAH, using Ficoll-Paque (Amersham Biosciences, Orsay, France) followed by Percoll density gradient centrifugation as previously described (13).
After total RNA extraction from monocytes, P-ECs, and PA-SMCs, reverse transcription was performed using random hexamer primers and reverse transcriptase (Invitrogen, Cergy-Pontoise, France). Primers for polymerase chain reaction were designed using Primer Express software (Applied Biosystems, Foster City, CA).
After protein concentration measurement, as described by Bradford (14), 100 μg of protein from each lung sample was used.
Cultures (5 · 104 cells/well) of P-ECs in MCDB131 and of PA-SMCs in Dulbecco's modified Eagle medium were prepared. The cultures were deprived of serum for 24 hours, after which the medium was collected for CCL2 assays. Freshly isolated monocytes (5 · 105 cells/ml) were resuspended in extraction buffer (phosphate-buffered saline, protease inhibitor cocktail, Triton 1%) and centrifuged at 1,800 rpm for 5 minutes at 4°C. The cell culture media were collected for CCL2 measurement. CCL2 levels in cell culture media and plasma were measured using an ELISA (R&D Systems, Lille, France) according to the manufacturer's instructions. Optical density at each appropriate wavelength was measured using a microplate reader (Molecular Devices, St. Grégoire, France). Values were expressed as picograms per 105 cells or picograms per milligram of lung tissue, and the means ± SEM were computed.
Immunohistochemistry was performed on 7-μm sections of frozen lung tissue. The sections were fixed, then processed with monoclonal mouse anti-human CCL2 (BD Pharmingen, le Pont-De-Claix, France).
The effect of EC-derived CCL2 on monocyte migration was measured using a modified Boyden's chamber (5-μm pore size, Transwell; Corning Costar Corporation, Badhoevedorp, The Netherlands).
PA-SMCs in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum were seeded in 24-well plates at a density of 5 · 104 cells/well and allowed to adhere as previously described (12). [3H]thymidine incorporated into the DNA was counted and expressed as counts per minute (cpm) per well.
PA-SMCs migration was assessed using a modified Boyden's chamber (Transwell; Corning Costar Corporation).
All data are reported as mean ± SEM. Analysis of variance (ANOVA) was used for between-group comparisons. When ANOVA indicated a significant difference, the groups were compared using a Mann-Whitney nonparametric test. P values less than 0.05 were considered statistically significant.
Plasma CCL2 concentrations were increased nearly twofold in patients with IPAH compared with control subjects (504 ± 36 vs. 317 ± 22 pg/ml, P = 0.002) (Figure 1A). Plasma CCL2 levels were not correlated with , PVR, or cardiac index. In a subgroup of nine patients studied before and after prostacyclin therapy, plasma CCL2 concentrations remained unchanged (446 ± 50 vs. 505 ± 60 pg/ml, respectively; P = 0.76) (Figure 1B). To determine whether the elevated circulating CCL2 concentration was related to increased CCL2 synthesis by monocytes, we measured the CCL2 content of monocytes from patients with IPAH and control subjects by ELISA. As shown in Figure 1C, no significant difference was found (295 ± 30 pg/106 cells from patients with IPAH and 212 ± 42 pg/106 cells from control subjects, P = 0.17).
Figure 1. (A) Circulating plasma CCL2 measurement using a specific sandwich immunoassay kit in 44 patients with idiopathic pulmonary arterial hypertension (IPAH) and 17 control subjects. Plasma CCL2 was elevated in patients with IPAH compared with control subjects (504 ± 36 vs. 317 ± 22 pg/ml, P = 0.002). Horizontal bars show the mean ± SEM (B) In a subgroup of 9 of 44 patients, circulating plasma CCL2 levels were not modified by initiation of continuous intravenous prostacyclin (PGI2) (446 ± 50 [before] vs. 505 ± 60 pg/ml [after], P = 0.76). (C) Monocyte CCL2 content was not significantly different between patients with IPAH (n = 4) and control subjects (n = 4) (295 ± 30 vs. 212 ± 42 pg/106 cells, P = 0.17). Each bar shows the mean ± SEM.
[More] [Minimize]CCL2 concentrations were increased in lung tissues from patients with IPAH compared with control subjects when assessed by Western blotting (Figure 2A) or ELISA (330 ± 82 vs. 112 ± 15 pg/mg total protein, P = 0.01). Immunohistochemical examination of lung specimens from patients with IPAH and control subjects showed preferential localization of CCL2 in perivascular infiltrated inflammatory cells and in endothelial cells (Figure 2B). No CCL2 immunostaining was detectable in the media layer of pulmonary vessels.
Figure 2. (A) Lung CCL2 immunoreactivity measured by Western blot in five patients with idiopathic pulmonary arterial hypertension (IPAH) and five control subjects. Each bar shows the mean ± SEM. CCL2 protein was increased in lung tissues from patients with IPAH compared with control subjects (1.84 ± 0.52 vs. 0.48 ± 0.06 arbitrary units, P = 0.009). (B) Immunolocalization of CCL2 in lungs from a patient with IPAH and a control subject. Muscular pulmonary arteriole from a patient with IPAH: note the pronounced perivascular infiltrate (a); muscular pulmonary arteriole from a patient with IPAH (b) and a control subject (c). Endothelial cells (arrows), wall-infiltrating cells, and alveolar macrophages (stars) react with anti-CCL2 antibody (red staining).
[More] [Minimize]CCL2 protein levels were markedly increased in media of P-ECs and PA-SMCs from patients with IPAH compared with control subjects (2,293 ± 50 pg/105 patient P-ECs vs. 623 ± 163 pg/105 control P-ECs, P = 0.02; and 1,278 ± 163 pg/105 patient PA-SMCs vs. 336 ± 129 pg/105 control PA-SMCs, P = 0.01). CCL2 levels in PA-EC media were about fourfold higher than those in PA-SMC media (Figure 3), both with cells from patients and with cells from control subjects. This difference was not related to the nuclear factor (NF)-κB signaling pathway, because phospho–NF-κB p65 protein levels did not differ between patients and control subjects (0.91 ± 0.10 vs. 0.92 ± 0.26 arbitrary units [AU] [P, nonsignificant] in cultured P-ECs and 0.52 ± 0.27 vs. 0.58 ± 0.26 AU [P, nonsignificant] in PA-SMCs, respectively).
Figure 3. CCL2 protein levels in media of quiescent pulmonary artery endothelial cells (P-ECs) and smooth muscle cells (PA-SMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) and control subjects. Each bar shows the mean ± SEM. (A) CCL2 levels were higher in media of P-ECs from patients with IPAH (n = 4) compared with control subjects (n = 4) (2,293 ± 50 vs. 623 ± 163 pg/105 cells, P = 0.02). (B) CCL2 levels were higher in the media of PA-SMCs from patients with IPAH (n = 6) compared with control subjects (n = 6) (1,278 ± 163 vs. 336 ± 129 pg/105 cells, P = 0.01).
[More] [Minimize]The presence of P-ECs in the Transwell lower chamber induced migration of monocytes from the upper to the lower chamber. This effect was more marked when P-ECs from patients with IPAH were used, compared with P-ECs from control subjects. In both cases, monocyte migration fell by 60% in the presence of CCL2-blocking antibody (P < 0.01). The antibody also markedly reduced the chemotactic effect of exogenous CCL2, whereas control antibody had no effect (P < 0.001). Interestingly, the chemotactic activity of P-EC media did not differ between cells from control subjects and cells from patients with IPAH when these last were tested in the presence of CCL2-blocking antibody (Figure 4).
Figure 4. Effects of endothelial cell–derived CCL2 on monocyte migration were studied using a modified Boyden's chamber. The mean number of pulmonary artery smooth muscle cells (PA-SMCs) from 10 randomly chosen high-power (×400) fields on the undersurface of the filter was computed. Pulmonary endothelial cells (P-ECs) or exogenous CCL2 (10 ng/ml) introduced into the lower chamber induced migration of monocytes from the upper to the lower chamber (*P < 0.01, **P < 0.001 vs. basal condition). This effect was more marked with P-ECs from patients with idiopathic pulmonary arterial hypertension (IPAH) than from control subjects (C). Monocyte migration was reduced by about 60% in the presence of CCL2-blocking antibody (CCL2 Ab) (#P < 0.01 vs. monocyte migration with P-ECs from patients or controls). Anti-CCL2 antibody also reduced the chemotactic effect of exogenous CCL2, whereas the antibody alone had no effect (§P < 0.001 vs. monocyte migration with CCL2). Each bar shows the mean ± SEM of four patients and four controls.
[More] [Minimize]CCL2 treatment of control PA-SMCs produced a concentration-dependent increase in [3H]thymidine incorporation, the maximal effect being observed with 10 ng/ml CCL2 (Figure 5A). Compared with values in control subjects, PA-SMC proliferation in response to CCL2 was more marked with cells from patients with IPAH (P < 0.05). Compared with values with cells under the basal condition (serum-free medium), the migration of PA-SMCs exposed to CCL2 increased in a concentration-dependent manner (Figure 5B), the maximal effect being achieved at 10 ng/ml. There was an increased migratory response to CCL2 of PA-SMCs from patients with IPAH compared with PA-SMCs from control subjects (Figure 5B).
Figure 5. (A) [3H]thymidine incorporation in cultured pulmonary artery smooth muscle cells (PA-SMCs) from five patients with idiopathic pulmonary arterial hypertension (IPAH) and five control subjects. The cells were incubated with increasing concentrations of CCL2 (0.1 to 10 ng/ml) in the presence of 0.3% fetal calf serum (FCS). Bars show the means ± SEM. *P < 0.05 versus basal condition, #P < 0.05 versus controls. (B) Migration of PA-SMCs from five patients with IPAH and five control subjects was investigated using a modified Boyden's chamber. The mean number of PA-SMCs from 10 randomly chosen high-power (×400) fields on the undersurface of the filter was computed. Compared with the basal condition (0% FCS), CCL2 stimulated PA-SMC migration in a concentration-dependent manner. The migratory response was slightly stronger with PA-SMCs from patients with IPAH than from control subjects. Bars show the means ± SEM. *P < 0.05 compared with the basal condition.
[More] [Minimize]In control subjects and patients with IPAH, CCR2 mRNA levels were higher in monocytes than in P-ECs or PA-SMCs. CCR2 mRNA levels were markedly increased in P-ECs and PA-SMCs from patients with IPAH compared with those from control subjects, whereas no differences in monocyte CCR2 mRNA concentrations were observed between patients with IPAH and control subjects (Figure 6).
Figure 6. CCR2 mRNA measured using quantitative reverse transcriptase–polymerase chain reaction in circulating blood monocytes, pulmonary endothelial cells (P-ECs), and pulmonary artery smooth muscle cells (PA-SMCs). In both control subjects and patients with idiopathic pulmonary arterial hypertension (IPAH), CCR2 mRNA expression was significantly higher in monocytes than in PE-Cs and PA-SMCs (*P < 0.0001 for controls; §P < 0.001 for patients with IPAH). CCR2 mRNA expression was significantly higher in P-ECs and PA-SMCs from patients with IPAH than from control subjects (#P < 0.0001 vs. P-ECs from controls; **P < 0.001 vs. PA-SMCs from controls). Each bar shows the mean ± SEM of four patients with IPAH and four control subjects.
[More] [Minimize]In our study, IPAH was associated with increased plasma and lung levels of CCL2, and P-ECs were a major source of CCL2 in the lung. In patients with IPAH, the increase in P-EC–derived CCL2 contributed substantially to the increased chemotaxis exerted by P-EC media on monocytes. Moreover, CCL2 induced mitogenic and chemotactic effects on PA-SMCs. These effects were stronger on PA-SMCs from patients with IPAH, due to increased expression of CCR2, the specific CCL2 receptor. Taken together, our results show that P-ECs from patients with IPAH constitutively synthesize excessive amounts of CCL2, which behaves as a chemoattractant for circulating inflammatory cells and as a growth factor for PA-SMCs. Thus, CCL2 may affect both the inflammatory process and the pulmonary vascular remodeling seen during progression of IPAH.
Endothelial activation, as occurs during atherosclerosis in response to oxidized lipids, leads to increased expression of chemokines and adhesion molecules, which induce recruitment of monocytes/lymphocytes into the subendothelium (15). During progression of PAH, increased lung expression of inflammatory cytokines coexists with accumulation of mononuclear inflammatory cells in the lungs and around the pulmonary vessels (3). The mechanisms that underlie these abnormalities are unknown. Although cytokines are secondary mediators of inflammation, they are not the primary triggers. In IPAH, the mechanisms by which inflammation develops and contributes to pulmonary vascular remodeling have not been elucidated.
In the present study, we focused on CCL2, which has been extensively studied in atherosclerosis and has been shown to act as a potent attractant of monocytes into the vessel wall. Experimental studies suggest a major role for CCL2 in the development of monocrotaline-induced PAH. Thus, in rats injected with monocrotaline, increases in serum and lung CCL2 levels antedated the development of pulmonary vascular remodeling and pulmonary hypertension (16). Plasma CCL2 levels were increased in patients with IPAH or chronic thromboembolic pulmonary hypertension, and some studies showed a positive correlation between CCL2 levels and PVR (17, 18).
In our study, plasma and lung CCL2 levels were elevated in patients with IPAH compared with control subjects, with no correlation between hemodynamic parameters and circulating CCL2 levels. Treatment with prostacyclin did not alter circulating CCL2 levels in a subgroup of nine patients. Interestingly, monocyte CCL2 content did not differ between patients with IPAH and control subjects, suggesting that the increased plasma CCL2 levels in IPAH did not originate in greater release from circulating monocytes. In contrast, the amount of CCL2 in media of cultured P-ECs and PA-SMCs from patients with IPAH was markedly elevated compared with that of control subjects. Our finding that CCL2 was expressed by both ECs and SMCs in the pulmonary circulation is consistent with previous studies of human cells from systemic blood vessels (18, 19). We found that the amount of CCL2 originating from ECs was 4 times greater than the amount from SMCs. Thus, ECs may be a major source of CCL2 in the lung. Results from patients with chronic thromboembolic pulmonary hypertension showing predominant immunostaining of CCL2 in the endothelium and neointima of large elastic pulmonary arteries support this possibility (17). A major finding from our study was that cultured cells from patients with IPAH overproduced CCL2. This overproduction did not seem to result from activation of the NF-κB pathway, because phospho–NF-κB p65 protein levels in cultured P-ECs and PA-SMCs did not differ between patients with IPAH and control subjects. Therefore, CCL2 overproduction may be a feature of the abnormal P-EC phenotype that likely contributes to the progression of IPAH.
To determine whether P-EC–derived CCL2 differentially affected monocyte recruitment in patients with IPAH and control subjects, we used double cell cultures to evaluate the migration of monocytes from healthy control subjects in the presence of P-ECs from patients with IPAH or from control subjects. We found that P-ECs were potent stimulators of monocyte migration, and that this effect was stronger with P-ECs from patients than from control subjects. Antibodies directed against CCL2 diminished the chemoattractant activity of P-EC medium by 60%, suggesting that CCL2 contributed substantially to monocyte migration. Thus, P-ECs from patients with IPAH constitutively release chemokines that exert chemoattractant effects on inflammatory cells. CCL2 may be the main chemokine involved in this process during IPAH.
Human vascular cells may be both sources and targets of cytokines. The effects of CCL2 on target cells are mediated by the CCR2 receptor, which is expressed by several cell types, including monocytes and SMCs (20, 21) CCL2 exerts mitogenic effects on human SMCs from systemic blood vessels and acts synergistically with many growth factors, including serotonin (22). We found that human PA-SMCs expressed the CCR2 receptor and that the level of expression was higher for cultured PA-SMCs from patients with IPAH than from control subjects. As a result, PA-SMCs from patients with IPAH grew faster than those from control subjects and showed a tendency toward increased migration in response to exogenous CCL2. These mitogenic and migratory effects of CCL2 on PA-SMCs are consistent with a direct role for CCL2 in pulmonary vascular remodeling. Increased CCR2 expression by PA-SMCs may also be a feature of the abnormal PA-SMC phenotype in IPAH. In previous reports, PA-SMCs from patients with PAH exhibited excessive proliferation in response to serotonin (5-hydroxytryptamine [5-HT]) or endothelin (ET)-1 but not to PDGF, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin growth factor (IGF), or transforming growth factor-β (12, 23). Moreover, PA-SMCs from patients with IPAH were less sensitive to growth inhibition by bone morphogenic proteins (BMPs) than were normal cells (24). Increased expression of the 5-HT transporter (12) and endothelin receptor ETA (25), as well as decreased expression of the BMP receptor type II (BMPRII) (26), were shown to mediate these altered responses. Our data extend these findings by showing that CCR2 overexpression is a feature of the abnormal PA-SMC phenotype in IPAH and results in increased cell stimulation by CCL2. Importantly, CCR2 expression by PA-SMCs from control subjects was negligible compared with that measured in monocytes. In patients with IPAH, however, CCR2 mRNA levels in PA-SMCs was one-third the mean value in monocytes. Moreover, increased CCR2 expression was also measured in P-ECs from patients with IPAH, suggesting that CCL2 autocrine loops in these cells, which may contribute to enhanced inflammation during progression of PAH.
The importance of CCL2 in PAH development was recently underlined by experimental animal studies showing that antisense oligonucleotides or anti-CCL2 antibodies protected against monocrotaline-induced PAH in rats (11, 16). Inflammation is known to be an important component of PAH in this model, raising the possibility that CCL2 may contribute to other forms of PAH. In recent studies, inflammation was shown to trigger development of PAH in BMPRII-deficient mice (27, 28). The present study in humans strongly supports a role for CCL2 overproduction in the pathogenesis of IPAH. The ability of CCL2 to both recruit inflammatory cells and act on PA-SMCs strongly suggests that CCL2 may be involved in both the inflammatory process and the pulmonary vascular remodeling that characterize IPAH.
The authors thank Ingrid Durand-Gasselin and Peter Dorfmüller for their technical assistance with the immunohistochemistry studies.
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