Abstract
Pulmonary vascular remodeling is key to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). We recently reported that fibroblast growth factor (FGF)2 is markedly overproduced by pulmonary endothelial cells (P-ECs) in IPAH and contributes significantly to smooth muscle hyperplasia and disease progression. Excessive FGF2 expression in malignancy exerts pathologic effects on tumor cells by paracrine and autocrine mechanisms.We hypothesized that FGF2 overproduction contributes in an autocrine manner to the abnormal phenotype of P-ECs, characteristic of IPAH. In distal pulmonary arteries (PAs) of patients with IPAH, we found increased numbers of proliferating ECs and decreased numbers of apoptotic ECs, accompanied with stronger immunoreactivity for the antiapoptotic molecules, B-cell lymphoma (BCL)2, and BCL extra long (BCL-xL) compared with PAs from control patients. These in situ observations were replicated in vitro, with cultured P-ECs from patients IPAH exhibiting increased proliferation and diminished sensitivity to apoptotic induction with marked increases in the antiapoptotic factors BCL2 and BCL-xL and levels of phosphorylated extracellular signal-regulated (ERK)1/2 compared with control P-ECs. IPAH P-ECs also exhibited increased FGF2 expression and an accentuated proliferative and survival response to conditioned P-EC media or exogenous FGF2 treatment. Decreasing FGF2 signaling by RNA interference normalized sensitivity to apoptosis and proliferative potential in the IPAH P-ECs. Our findings suggest that excessive autocrine release of endothelial-derived FGF2 in IPAH contributes to the acquisition and maintenance of an abnormal EC phenotype, enhancing proliferation through constitutive activation of ERK1/2 and decreasing apoptosis by increasing BCL2 and BCL-xL.
Pulmonary arterial hypertension (PAH) is a disease in which obstructive remodeling of the pulmonary arteries (PAs) leads to progressive elevation of pulmonary vascular resistance (PVR) causing an impedance of right ventricular outflow, resulting in dyspnea, fatigue, chest pain, and syncope (1). One of the most severe forms is idiopathic PAH (IPAH), which is characterized by a marked increase in PVR, eventuating in progressive right heart failure and death within 5 years after the diagnosis (1, 2). The pulmonary vascular remodeling of PAH includes concentric medial thickening of the distal PAs, neo-muscularization of previously nonmuscular capillary-like vessels, structural wall changes in larger PAs, and the development of complex vascular structures known as plexiform lesions. The remodeling process involves all three vessel wall layers (adventitia, media, and intima) and all vessel wall cell types (endothelial cells [ECs], smooth muscle cells [SMCs], fibroblasts, inflammatory cells, and platelets). However, although the histopathology of IPAH has been extensively documented, the mechanisms underlying the progressive obliteration of the distal PAs remain incompletely understood (3–5).
ECs play a central role in vascular homeostasis, and recent research has established that endothelial dysfunction contributes to the structural changes that characterize PA remodeling (6–10). Loss of endothelial-peroxisome proliferator-activated receptor type γ (PPARγ) in mice results in muscularization of PA and PAH (8). Our group has shown that pulmonary ECs from patients with IPAH release excessive amounts of soluble growth factors able to act on SMCs (6, 7, 9). Paracrine overproduction of serotonin, endothelin-1 (6), and fibroblast growth factor (FGF)2 (9) contribute to the increased SMC proliferation that characterizes PAH.
Our recent findings highlight a major role for FGF2 in the cross talk between P-ECs and SMCs in the pathobiology of PAH. FGF2 is markedly overproduced by P-ECs in IPAH and contributes significantly to SMC hyperplasia and disease progression in humans and rodents (9). FGFs have been implicated in the proliferation, migration, survival, and differentiation of many cell types, including ECs (11–14). Accentuated FGF2 expression has been documented in malignancies, whereby FGF2 acts on tumor cells via paracrine or autocrine mechanisms (15). Furthermore, FGF2 binds heparan sulfate proteoglycans and heparin, thereby establishing a biological reservoir that can be released locally in a regulated manner (16, 17). This suggests that, in addition to the paracrine effect recently demonstrated in the PA-SMC, the excess FGF2 observed in PAH might modulate the P-EC phenotype via separate autocrine effects.
Therefore, we hypothesized that autocrine overproduction of EC-derived FGF2 contributes to the acquisition and maintenance of a hyperproliferative and apoptosis-resistant phenotype in P-ECs in patients with IPAH.
This study was approved by the local ethics committee (CPP Ile-de-France, Le Kremlin-Bicêtre, France). All patients gave informed consent before the study.
Lung specimens were obtained at the time of transplantation from 10 patients with IPAH (Table 1) at the Marie Lannelongue Hospital, Le Plessis-Robinson, France. Patients with mutations in the bone morphogenic protein-receptor II (BMPRII) or activin-like kinase-type 1 (ALK-1) genes were excluded. Control lung specimens were obtained from patients without evidence of pulmonary vascular disease who underwent lobectomy or pneumonectomy for localized lung cancer, with the normal tissue collected at a distance from the tumors.
Characteristics | Control Patients | IPAH Patients |
|---|---|---|
| Male/female | 3/7 | 3/7 |
| Age, yr, median (range) | 41 (20–54) | 34 (18–47) |
| NYHA functional class III | NA | 2 |
| NYHA functional class IV | NA | 8 |
| mPAP, mm Hg | NA | 60 ± 4 |
| PVR, dyne/s/cm5 | NA | 1,130 ± 136 |
Human P-ECs were isolated and cultured as previously described (7). For apoptosis induction, P-ECs were placed in serum-free medium for 24 or 48 hours or incubated for 16 hours in medium with 0.5% FCS in presence of cycloheximide, H2O2, or vehicle. Additional studies were performed in the presence of human recombinant FGF2 (rHu-FGF2) (Sigma-Aldrich, Saint-Quentin Fallavier, France) conditioned media (diluted in serum-free media; 2:3) from control or IPAH P-ECs, PD98059 (25 μM) (Sigma-Aldrich), or FCS in the concentrations stated in the legends.
To suppress BCL2, BCL-xL, or FGF2 expression, P-ECs were transfected using lipofectamine RNAiMAX with 100 nM of BCL2-, BCL-xL-, or FGF2 siRNA or with a scrambled sequence (Invitrogen, Cergy-Pontoise, France). The cells were studied within 3 days after transfection. Suppression of BCL2, BCL-xL, or FGF2 levels was documented 72 hours after transfection.
Floating cells were collected and combined with adherent cells harvested by trypsin/EDTA treatment and then stained with the Annexin V-FITC (BD Biosciences, Pont-de-Claix, France). In each sample, at least 10,000 cells were counted by FACS analysis.
EC proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation and by cell counting. BrdU staining was measured by the DELFIA Cell proliferation kit (PerkinElmer, Courtaboeuf, France) and a time-resolved fluorometer EnVisionTM Multilabel Reader (PerkinElmer).
Cells were counted using a Neubauer hemocytometer (American Optical Company, Buffalo, NY) with trypan blue dye exclusion.
The mRNA expression of Bax, BCL2, BCL-xL, FGF2, and FGF receptors was measured by real-time quantitative PCR as previously described (18).
P-ECs were homogenized and sonicated in PBS containing protease and phosphatase inhibitors (Sigma-Aldrich). Protein extract (50 μg) was used to detect BCL2, BCL-xL, phosphoERK1/2, ERK2 (1:250) (Tebu-Bio, Le Perray-en-Yvelines, France), or Bax protein (1:1,000) (Euromedex, Mundolsheim, France) by SDS-PAGE was performed as previously described (18). FGF2 protein levels were measured in the conditioned media using an ELISA kit (R&D Systems, Lille, France). For immunochemistry, paraffin sections were incubated overnight at 4°C with antibodies to BCL2, BCL-xL (1:50), von Willebrand factor (vWF), or proliferating cell nuclear antigen (PCNA; 1:200; Dako, Trappes, France) as previously described (18). We conducted terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) using the ApopTag red in situ apoptosis detection kit (Chemicon, Molsheim, France) (19).
Values of each variable are expressed as mean ± SEM. Statistical significance was tested using the nonparametric Mann-Whitney test or two-way ANOVA followed by the Student-Newman-Keuls test. Values were considered statistically significant at P < 0.05.
To determine the degree of EC apoptosis in the lungs of patients with IPAH, we performed immunohistochemistry on lung specimens from 10 patients with IPAH and 10 control subjects (Table 1). Coimmunostaining with antibodies against PCNA and vWF showed that IPAH specimens contained 2 to 3 times more proliferating ECs in distal PAs (Figure 1A and Figures E1 and E2 in the online supplement). Double staining for TUNEL and vWF showed a 2-fold decrease in the number of apoptotic ECs in distal PAs compared with the control specimens (Figure 1B and Figures E1 and E3).
Figure 1. Representative photomicrographs of distal pulmonary arteries (PAs) in lung sections from patients with idiopathic pulmonary arterial hypertension (IPAH) and control subjects. Coimmunolocalization of (A) von Willebrand factor (vWF) and proliferating cell nuclear antigen (PCNA), (B) vWF and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), (C) vWF and B-cell lymphoma (BCL)2, and (D) vWF and B-cell lymphoma–extra long (BCL-xL). The percentages of PCNA- and TUNEL-positive ECs were obtained from the analysis of 15 to 20 different distal pulmonary arteries selected randomly in each patient. Arrows: PCNA- or TUNEL-positive pulmonary endothelial cells (P-ECs). *PCNA- or TUNEL-positive cells. Scale bar = 50 μm. Values are mean ± SEM (n = 3–5). **P < 0.01 and ***P < 0.001 compared with control PAs.
[More] [Minimize]The balance between pro- and antiapoptotic factors determines the amount of apoptosis. Therefore, we conducted parallel immunohistochemical evaluations to investigate whether the expression of antiapoptotic family members was modified in distal PA endothelium from patients with IPAH. Consistent with the in situ incidence of EC apoptosis, we found strong BCL2 (Figure 1C) and BCL-xL (Figure 1D) staining within the endothelium of distal PAs from patients with IPAH as compared with a weak staining within the endothelium of control specimens.
To determine whether the abnormal phenotype of P-ECs from patients with IPAH found in situ was maintained in vitro, primary P-EC cultures were generated from the lung specimens and used to evaluate the proliferation/cell death balance under basal conditions and after apoptosis induction by serum deprivation.
Under basal conditions (0.5% FCS), proliferation of IPAH P-ECs was increased by approximately 35% compared with control–P-ECs (Figure 2A). In addition, FCS increased P-EC proliferation in a dose-dependent manner, and this effect was even more pronounced with cells from patients with IPAH (Figures 2A and 2B). Furthermore, although cell proliferation decreased after 24 hours without serum, the difference between control P-ECs and IPAH P-ECs was maintained. We next extended these results using fluorescence-activated cell sorting (FACS) to analyze and compare the cell cycle between control and IPAH P-ECs. With 10% FCS, control versus IPAH P-ECs were distributed respectively as follows: subG0 phase, 13.8 ± 1.2% versus 6.8 ± 1.7%, P < 0.05; G0/G1 phase, 72.1 ± 1.5% versus 72.6 ± 4.4%, NS; and S+G2/M phase, 14.1 ± 1.1% versus 20.6 ± 1.9%, P < 0.05. Serum deprivation for 24 hours led to a marked increase in subG0-phase cells among control P-ECs but not IPAH P-ECs (Figure E4).
Figure 2. In vitro proliferation and apoptosis analysis of human P-ECs isolated from IPAH and control lung biopsies. Proliferation of control- and IPAH P-ECs was assessed by (A) 5-bromo-2-deoxyuridine (BrdU) incorporation and (B) viable cell counts under basal conditions in the presence of FCS or after apoptosis induction by serum deprivation (24 h). (C) Representative two-color FACS analysis of the annexinV-FITC assays and quantification of the percentage of annexinV+/PI− P-EC under basal conditions in the presence of FCS and after apoptosis induction by serum deprivation. (D) Evaluation of sensitivity to apoptosis induced by H2O2 and cycloheximide. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control P-ECs under basal conditions. §P < 0.05, §§P < 0.01, and §§§P < 0.001 between control and IPAH P-ECs subjected to the same treatment.
[More] [Minimize]In parallel experiments, we used annexinV FITC/PI staining and flow cytometry analysis to look for differences in apoptosis between control and IPAH P-ECs with and without serum (Figure 2C). No significant difference in the percentage of apoptotic cells (annexinV+/PI−) was noted between control and IPAH P-ECs under basal conditions. After serum deprivation for 24 hours, we found a significant increase in the percentage of annexinV+/PI− cells among control P-ECs compared with IPAH P-ECs. After 48 hours of serum deprivation, annexinV+/PI− cells contributed greater than 50% of control P-ECs and greater than 25% of IPAH P-ECs.
We used other apoptosis-inducing stimuli to further explore the sensitivity of IPAH P-ECs to apoptosis compared with control–P-ECs (Figure 2D). Consistent with the results obtained after serum deprivation, we found that IPAH–P-ECs were less sensitive than control P-ECs to apoptosis induction by H2O2 or cycloheximide.
Subsequent studies were performed to investigate whether the expression of genes for pro- and antiapoptotic factors differed between control and IPAH–P-ECs. First, we measured mRNA levels of Bax, BCL2, and BCL-xL (Figure 3A). No significant differences were found in the mRNA expression of the proapoptotic factor Bax. In contrast, IPAH P-ECs had significantly higher levels of mRNA for antiapoptotic molecules BCL2 and BCL-xL as compared with control P-ECs. These results suggested an imbalance with an excess of antiapoptotic factors over proapoptotic factors in IPAH P-ECs compared with control P-ECs. Consistent with the gene expression results, no differences in Bax protein levels were found between the two groups (Figure 3B). However, BCL2 and BCL-xL proteins were significantly increased in IPAH P-ECs as compared with control P-ECs. Under basal conditions, IPAH P-ECs had an approximately 35% decrease in the Bax:BCL2 ratio and an approximately 40% decrease in the Bax:BCL-xL ratio compared with control P-ECs.
Figure 3. Evaluation of the expression of proapoptotic (Bax) and antiapoptotic (BCL2 and BCL-xL) factors and ERK1/2 phosphorylation status in cultured human control and IPAH P-ECs and role in the apoptosis resistance and the proliferative potential. (A) Relative levels of mRNAs encoding Bax, BCL2, and BCL-xL and values of the Bax:BCL2 and Bax:BCL-xL ratios. (B) Representative Western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control and quantification of the Bax:BCL2 and Bax:BCL-xL ratios. (C) Representative Western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control in IPAH P-ECs 72 hours after transfection with lipofectamine alone, scrambled sequence, BCL2 siRNA, or BCL-xL siRNA. (D) Quantification of the percentage of annexinV+/PI− cells in the IPAH P-EC population with 10% FCS or after apoptosis induction by serum deprivation (24 h) 48 hours after transfection with BCL2 siRNA and/or BCL-xL siRNA or scrambled sequence. (E) Representative Western immunoblots of phosphoERK1/2 and ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. (F) Proliferation of control and IPAH P-ECs assessed by BrdU incorporation under basal condition in presence of PD98059 or vehicle. Values are mean ± SEM (n = 5–10). *P < 0.05, **P < 0.01, and ***P < 0.001 between control and IPAH P-ECs or compared with IPAH P-ECs transfected with the scrambled sequence. §P < 0.05 between control and IPAH P-ECs subjected to the same treatment.
[More] [Minimize]Next, we investigated whether decreasing BCL2 and BCL-xL levels in cultured human IPAH P-ECs by RNA interference restored sensitivity to apoptosis induction. Transfecting P-ECs with BCL2 siRNA and BCL-xL siRNA decreased the BCL2 protein level by about 87% and the BCL-xL protein level by about 80% compared with transfection with scrambled siRNA (Figure 3C). Compared with scrambled sequence, BCL2 siRNA or BCL-xL siRNA markedly increased the level of apoptosis induced by serum deprivation of IPAH P-ECs (Figure 3D). In the presence of serum, treatments of IPAH P-ECs with the scrambled sequence or BCL2 siRNA were associated with similar basal levels of apoptosis. In contrast, BCL-xL siRNA induced a 3-fold increase in the basal level of IPAH P-EC apoptosis (Figure 3D).
We next explored whether the hyperproliferative potential of IPAH P-ECs was due to changes in the basal ERK1/2 phosphorylation status. ERK1/2 are members of the MAPK superfamily known to be a potent regulator of cell growth that can be activated in response to a diverse range of extracellular stimuli, including mitogens, growth factors, and cytokines (20). First, we used Western blot analysis to assess the basal ERK1/2 phosphorylation status and found a constitutive activation of ERK1/2 in IPAH P-ECs as compared with control P-ECs (Figure 3E). Second, we showed that inhibition of ERK1/2 activation by the selective antagonist of MEK (MAPK/ERK kinase) PD98059 almost completely normalized the proliferation of IPAH P-ECs (Figure 3F). Taken together, these results strongly suggest that the constitutive activation of ERK1/2 and the hyperproliferative phenotype of IPAH P-ECs are connected.
Although many genes may contribute to the resistance to apoptosis, previous studies have demonstrated that FGF2 promotes resistance to apoptosis in NIH-3T3 cells, chronic lymphocytic leukemia B-cell lines, and bovine aortic ECs (11–14). Inappropriate expression of FGF2 and its receptors causes aberrant cell proliferation in many cancers (21). Furthermore, we recently reported that endothelial FGF2 was overproduced by IPAH P-ECs as compared with control P-ECs in vitro and in vivo (9). Thus, to investigate whether FGF2 signaling was enhanced in the IPAH P-ECs not only via increased FGF2 expression but also due to alterations in FGF2 receptors expression, we measured the mRNA levels of FGF-R1, FGF-R2, FGF-R3, and FGF-R4 (Figure 4A). No differences in mRNA levels of FGF-R1, FGF-R3, or FGF-R4 were found. In contrast, the mRNA (Figure 4A) and protein (Figure 4B) expression of FGF-R2 was substantially increased in IPAH P-ECs compared with control P-ECs. Consistent with our previous findings (9), we also found that FGF2 was markedly overproduced in IPAH P-ECs as compared with control P-ECs at the mRNA (Figure 4A) and protein levels (13.3 ± 2.7 versus 2.6 ± 0.9 pg/ml in conditioned media by ELISA, respectively; P < 0.001).
Figure 4. Analysis of mRNA levels of fibroblast growth factor (FGF)2 and its receptors in cultured human control and IPAH P-ECs and evaluation of their responsiveness to FGF2 under serum deprivation. (A) Relative levels of mRNAs encoding FGF2 and FGF-Rs in control and IPAH P-ECs. (B) Representative Western immunoblots of FGF-R2 and β-actin loading control and quantification of the FGF-R2:β-actin ratio. (C) Evaluation of the balance between proliferation/apoptosis of quiescent control and IPAH P-ECs under serum deprivation with or without conditioned media from IPAH or control P-ECs (D) or rHu-FGF2. Bars represent mean ± SEM (n = 4–8). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control P-ECs. §P < 0.05 and §§P < 0.01 between control and IPAH P-ECs subjected to the same treatment.
[More] [Minimize]We then investigated the responsiveness of control and IPAH P-ECs to conditioned media from either cell population under serum deprivation (Figure 4C). Compared with control P-EC media, IPAH P-EC media induced greater proliferation of cultured control P-ECs. The proliferation induced by the two media was even more pronounced when cultured IPAH P-ECs were used. In parallel experiments, we used annexinV FITC/PI staining and flow cytometry analysis to evaluate potential protective effects of conditioned control and IPAH P-EC media on serum deprivation–induced apoptosis of control and IPAH P-ECs. The level of induced apoptosis of cultured control P-ECs was lower with IPAH P-EC media than with control P-EC media. No significant changes in the percentage of annexinV+/PI− cells were observed with the use of IPAH P-ECs.
We then evaluated the responsiveness of control and IPAH P-ECs to rHu-FGF2 under serum deprivation (Figure 4D). The proliferation and survival of control P-ECs were increased dose-dependently by rHu-FGF2, and this effect was even more pronounced on IPAH P-ECs. Treatment with rHu-FGF2 in a dose of 0.1 ng/ml offered significant protection from serum deprivation–induced apoptosis.
Previous studies demonstrated that FGF2 promotes resistance to apoptosis in many types of differentiated cells and that this effect correlated with increased expression of BCL2 and BCL-xL (11–14). Based upon our immunohistochemical findings and our gene expression results showing marked increases in BCL2 and BCL-xL levels in IPAH P-ECs, we hypothesized that the antiapoptotic properties of FGF2 in P-ECs were mediated chiefly by up-regulation of these antiapoptotic proteins. To test this hypothesis, we first evaluated whether FGF2 modulated their expression in our primary cultures. Using control P-ECs treated with increasing rHu-FGF2 doses in the absence of serum, we found no changes in mRNA levels of Bax protein levels compared with control P-ECs treated with vehicle. In contrast, BCL2 and BCL-xL protein levels increased dose dependently in response to rHu-FGF2 compared with control P-ECs treated with vehicle (Figure 5A). We found that 10 ng/ml of rHu-FGF2 significantly decreased the Bax/BCL2 and Bax/BCL-xL ratios by approximately 30%.
Figure 5. Effect of FGF2 on the expression of Bax, BCL2, or BCL-xL on the ERK1/2 phosphorylation status and its autocrine contribution to protection against serum-deprivation–induced apoptosis and to the excessive proliferation. (A) Representative Western immunoblots of Bax, BCL2, BCL-xL, and β-actin loading control and quantification of the Bax:BCL2 and Bax:BCL-xL ratios. (B) Representative Western immunoblots of phosphoERK1/2 and ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. (C) FGF2 protein levels in conditioned media from control and IPAH P-ECs with or without FGF2 siRNA or scrambled sequence. (D) Representative two-color FACS analysis of the annexinV-FITC assays and quantification of the percentage of annexinV+/PI− cells in control P-EC and IPAH P-EC populations after apoptosis induction by serum deprivation (24 h) 48 hours after transfection with FGF2 siRNA or scrambled sequence with or without rHu-FGF2 (10 ng/ml). (E) Representative Western immunoblots of Bax, BCL2, and BCL-xL and quantification of the Bax:BCL2 and Bax:BCL-xL ratios. (F) Proliferation of control and IPAH P-ECs 48 hours after transfection with FGF2 siRNA or scrambled sequence assessed by BrdU incorporation. (G) Representative Western immunoblots of phosphoERK1/2 and ERK2 and quantification of the phosphoERK1/2:ERK2 ratio. Values are mean ± SEM (n = 3–7). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with 0 ng/ml of rHu-FGF2 or with control P-ECs transfected with the scrambled sequence. §P < 0.05 between control and IPAH P-ECs subjected to the same treatment.
[More] [Minimize]We then assessed and confirmed that FGF2 signaling is a potent modulator of ERK phosphorylation and activation in P-ECs by showing that rHu-FGF2 application (10 ng/ml) led to a time-dependent increase in ERK1/2 phosphorylation (Figure 5B).
We next investigated the contribution of autocrine FGF2 to protection against serum deprivation–induced apoptosis of human IPAH P-ECs. We determined if decreasing FGF2 by RNA interference would restore sensitivity to apoptosis induction. We first measured soluble FGF2 levels in conditioned media by ELISA and found that FGF2 siRNA decreased FGF2 protein levels in control P-ECs and IPAH P-ECs (Figure 5C). In IPAH P-ECs, FGF2 siRNA decreased the FGF2 protein level by 78% as compared with the scrambled sequence. Consistent with our previous findings, after transfection with scrambled siRNA, the percentage of annexinV+/PI− cells was significantly lower among IPAH P-ECs than among control P-ECs after 24 hours of serum deprivation (Figure 5D). In contrast, IPAH P-ECs transfected with FGF2 siRNA contained a substantially higher proportion of annexinV+/PI− cells compared with IPAH P-ECs transfected with the scrambled sequence at the same time point (Figure 5D). Furthermore, the percentage of annexinV+/PI− cells was similar among IPAH P-ECs transfected with FGF2 siRNA and control P-ECs transfected with the scrambled sequence. In addition, the apoptosis resistance of IPAH P-ECs transfected with FGF2 siRNA was restored in the presence of rHu-FGF2, compared with IPAH P-ECs transfected with FGF2 siRNA alone (Figure 5D). Consistent with our hypothesis, we found that this restoration of sensitivity to apoptosis in IPAH P-ECs was related to decreased expression of BCL2 and BCL-xL proteins (Figure 5E).
In many cancers, an autocrine FGF activation loop controls ERK1/2 activation (21). Therefore, we investigated whether excessive autocrine release of endothelial-derived FGF2 explained the constitutive ERK1/2 activation and thus the hyperproliferative phenotype of IPAH P-ECs. To test this theory, we used BrdU and Western blot analysis to assess the effect of FGF2 depletion on EC proliferation and ERK1/2 phosphorylation status. Under basal conditions, FGF2 depletion in IPAH P-ECs by RNA interference almost completely abolished the excessive proliferation and the constitutive ERK1/2 activation in IPAH P-ECs compared with IPAH P-ECs transfected with the scrambled sequence (Figures 5F and 5G). Collectively, these results strongly suggest that excessive autocrine release of endothelial-derived FGF2 may contribute to the hyperproliferative phenotype through constitutive activation of ERK1/2.
We report evidence that autocrine FGF2 signaling is critical for the acquisition and maintenance of a hyperproliferative and apoptosis-resistant phenotype in pulmonary ECs in IPAH. Muscularized walls of distal PAs from IPAH lung specimens contained large numbers of proliferating ECs (a 2- to 3-fold increase) and small numbers of apoptotic ECs (a 2-fold decrease), as well as increased immunoreactivity for BCL2 and BCL-xL, compared with control lung specimens. These in situ findings were replicated in vitro: Cultured human IPAH P-ECs exhibited a hyperproliferative and apoptosis-resistant phenotype compared with control P-ECs. With three different apoptosis-inducing stimuli, IPAH P-ECs were less sensitive to apoptosis induction compared with control P-ECs. We then showed that this apoptosis-resistant phenotype of IPAH P-ECs was mediated by an excess of antiapoptotic factors over proapoptotic factors, with increased expression of BCL2 and BCL-xL mRNA and proteins as compared with control cells. In addition, we obtained evidence that the hyperproliferative phenotype of IPAH P-ECs was associated with constitutive activation of ERK1/2. Finally, we demonstrated that this abnormal phenotype resulted from an autocrine loop of EC-derived FGF2 in IPAH P-ECs. These findings provide further evidence of the multifunctional role for FGF2 signaling in the pathophysiology of IPAH and support the notion that FGF2 may be a promising new treatment target in the disease.
Previous in vitro and animal studies have suggested that the balance between EC proliferation/apoptosis is disturbed in IPAH, but the exact nature of these defects remains to be elucidated (10). Our in situ immunohistochemical studies of the distal PA endothelium from patients with IPAH showed a 2- to 3-fold increase in proliferating ECs and a 2-fold decrease in apoptotic ECs, as compared with control subjects. Thus, abnormal regulation of the balance between proliferation and apoptosis appears to be present even in the late stages of the disease. Although this balance is vital for vascular wall homeostasis and remodeling, the mechanisms that coordinate apoptosis and proliferation remain incompletely understood. We found strong BCL2 and BCL-xL staining in the distal PA endothelium of patients with IPAH as compared with control subjects, suggesting that the decrease in apoptotic ECs reflects an excess of antiapoptotic factors over proapoptotic factors. The abnormalities found in situ persisted in vitro. First, cultured IPAH P-ECs exhibited a hyperproliferative phenotype and diminished sensitivity to apoptosis induction compared with control P-ECs. Consistent with our results obtained in P-ECs, Masri and colleagues reported that pulmonary artery ECs from patients with IPAH exhibited an unusual hyperproliferative potential, with decreased susceptibility to apoptosis (22). Second, we found similar Bax mRNA and protein levels but markedly increased BCL2 and BCL-xL mRNA and protein levels in cultured IPAH P-ECs compared with control P-ECs. Our results demonstrate that an excess of antiapoptotic factors over proapoptotic factors likely accounts for the decreased sensitivity to apoptosis induction observed in the IPAH P-ECs in vitro. Indeed, IPAH P-EC treatment with BCL2 siRNA caused a 3-fold increase in sensitivity to apoptosis compared with IPAH P-EC treatment with the scrambled sequence. IPAH P-EC treatment with BCL2 siRNA did not affect the basal apoptotic level in the presence of serum. However, P-EC treatment with BCL-xL siRNA markedly increased not only IPAH P-EC sensitivity to apoptosis but also the basal apoptotic level in the presence of serum. These findings are consistent with a recent report by Ackermann and colleagues showing that, in human umbilical vein ECs, inhibition of endogenous BCL-xL production by antisense oligonucleotides caused 10 to 25% of the cell population to undergo apoptosis under basal conditions (23). One possible explanation is that BCL-xL, but not BCL2, plays a primary role in basal EC viability. Alternatively, qualitative and quantitative differences in the functional activity of these two antiapoptotic proteins may explain these differences. Fiebig and colleagues recently reported that BCL-xL was qualitatively different from and 10 times more effective than BCL2 under the same conditions (24). It has also been reported that BCL2 and BCL-xL can differentially block chemotherapy-induced cell death, with BCL-xL showing a stronger protective effect than BCL2 against cell death induced by several chemotherapeutic agents (25). In addition, we obtained evidence that constitutive activation of ERK signaling in IPAH P-ECs contributes to the hyperproliferative phenotype. Taken together, our results constitute strong evidence that the abnormal hyperproliferative apoptosis-resistant IPAH P-EC phenotype is ascribable to an intrinsic abnormality present in these cells. Because the tissue sections and cells we obtained were from patients with end-stage disease receiving drug therapy, we cannot exclude the possibility that these abnormalities represent a compensatory mechanism and do not participate in early-stage vascular remodeling in IPAH. However, several recent reports suggest that proapoptotic treatments used in cancer (including dichloroacetate, imatinib, and antisurvivin agents) may reverse pulmonary vascular remodeling and PAH (26–28). Despite the importance of EC apoptosis, the precise molecular mechanism underlying the regulation of apoptotic pathways remains largely undetermined.
Although apoptosis is a multistage, genetically controlled process that allows normal cell turnover, it is subject to physiologic and pathophysiologic regulation (29) mediated in part by dependence upon growth factors and cytokines (30–33). We recently reported that FGF2 was overproduced by IPAH P-ECs and contributed to SMC hyperplasia and to IPAH progression in humans and rodents (9). FGF2 is a multifunctional cytokine involved in many biological processes, including cell proliferation, differentiation, migration, neoangiogenesis, and cell survival. Recent work has shown that FGFs, including FGF2, can promote resistance to chemotherapeutic agents in vitro and in vivo (34). This is of interest not only because FGF2 is overproduced in patients with IPAH but also because the extracellular matrix (ECM) acts as a physiological reservoir for secreted FGF2, and various enzymes (including metalloproteinases, plasmin, and urokinase-type plasminogen) are capable of releasing ECM-bound FGF2. This delicate balance between FGF2 storage and release in the ECM probably modulates the biological effects of FGF2 on the endothelium. P-EC treatment with FGF2 siRNA almost completely restored sensitivity to apoptosis induced by serum deprivation, whereas no change was observed with the scrambled sequence. This phenomenon was strongly related to decreased expression of BCL2 and BCL-xL proteins and was abolished in the presence of rHu-FGF2. In addition, under basal conditions, FGF2 depletion by RNA interference almost completely abolished the excessive proliferation and the constitutive ERK1/2 activation in IPAH P-ECs compared with IPAH P-ECs transfected with the scrambled sequence. Collectively, these findings indicate that increased activity of the FGF2 autocrine loop is among the mechanisms needed to acquire not only the in vitro P-EC apoptosis-resistant phenotype but also the in vitro P-EC hyperproliferative phenotype.
We previously reported that the conditioned media from IPAH P-ECs did not contain greater amounts of PDGF, EGF, and TGF-β as compared with control P-EC media, suggesting that these growth factors were not involved in the acquisition and maintenance of the hyperproliferative apoptosis-resistant phenotype (9). In contrast, the amount of FGF2 secreted into the media by IPAH P-ECs was five times greater than the amount released by control P-ECs. We cannot exclude the involvement of other growth factors, such as insulin growth factor-1 (35), vascular endothelial growth factor (36, 37), or bone morphogenetic proteins (38), to support EC growth and prevent EC apoptosis. However, multiple lines of evidence suggest that FGF2 contributes significantly to the acquisition and maintenance of the abnormal P-EC phenotype. First, FGF2 binding to the ECM leads to underestimation of the amount of FGF2 released by IPAH P-ECs and also concentrates active FGF2 within the ECM in close proximity to the P-ECs. Second, FGF2 knockdown in IPAH P-ECs returns not only the sensitivity to apoptosis but also the proliferative potential to a level close to that of control P-ECs. Herein, we obtained evidence for altered responsiveness to rHu-FGF2 and to conditioned media in IPAH P-ECs when compared with control P-ECs, suggesting that dysregulation of the FGF2 signaling pathway was associated not only with FGF2 overproduction but also with altered expression of FGF receptors. Consistent with our hypothesis, we found increased FGF-R2 expression in IPAH P-ECs as compared with control P-ECs. Matsunaga and colleagues (39) recently demonstrated that overexpression of a constitutively active FGF-R2 in ECs in vitro enhanced migration and survival and augmented autocrine FGF2 production. Therefore, increased FGF-R2 protein/activity in IPAH P-ECs may contribute to the excessive FGF2 secreted by these cells. Deficient activity of PPARγ may also explain the augmented FGF2 production by IPAH P-ECs. Loss of PPARγ is associated with the development of PAH (8, 40) and the modulation of a number of endothelial genes, including up-regulation (41) of FGF2. It is also possible that ECs overexpressing FGF2 are selected early in the pathogenesis of IPAH (10), thus contributing to the expansion of a hyperproliferative apoptosis-resistant population.
FGF-R2, similar to the other FGF-Rs, is a specific split tyrosine kinase receptor coupled to multiple intracellular signaling pathways, including the MAPK pathway, the phospholipase-C/protein kinase-C pathway, and the phosphatidylinositol 3-kinase/Akt- and Src-associated pathways (15). Tyrosine kinase receptor activation by growth factors enhances cell survival through several mechanisms, including regulation of the BCL2 family proteins (30–33). However, the molecular mechanisms by which FGF2 promotes resistance to apoptosis in human P-ECs are incompletely understood. Here, we showed that (1) FGF2 treatment led to rapid and marked increases in BCL2 and BCL-xL and to ERK1/2 phosphorylation and activation in primary cultures of human P-ECs and (2) inhibition of the autocrine FGF2 production in IPAH P-ECs restores sensitivity to apoptosis induced by serum deprivation and normalizes the excessive proliferation under basal condition.
In summary, this study underscores a novel mechanism by which endothelial FGF2 overproduction contributes in an autocrine manner not only to the apoptosis-resistant phenotype but also to the hyperproliferative phenotype of P-ECs, both of which are characteristic of IPAH. The apoptosis-resistant phenotype was directly mediated by an imbalance of proapoptotic over antiapoptotic factors. We obtained evidence that the constitutive activation of ERK1/2 contributes to the hyperproliferative phenotype of IPAH P-ECs. We show that FGF2 modulates not only the expression of some of these antiapoptotic factors in human P-ECs, including BCL2 and BCL-xL, but also ERK phosphorylation and activation in human P-ECs. Collectively, our data support the notion that the overproduction of EC-derived FGF2 in IPAH contributes to multiple different components of the disease, including SMC hyperplasia via a paracrine effect and the acquisition and maintenance of a hyperproliferative apoptosis-resistant phenotype via an autocrine loop. Taken together with data demonstrating a beneficial effect in several malignant disorders and from our previous study in an animal model of PAH, these results suggest that the FGF2 signaling pathway may represent an attractive therapeutic target in PAH.
| 1. | Galie N, Hoeper MM, Humbert M, Torbicki A, Vachiery JL, Barbera JA, Beghetti M, Corris P, Gaine S, Gibbs JS, et al. Guidelines for the diagnosis and treatment of pulmonary hypertension. Eur Respir J 2009;34:1219–1263. |
| 2. | Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, Elliott CG, Gaine SP, Gladwin MT, Jing ZC, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2009;54:S43–S54. |
| 3. | Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones PL, Maitland ML, Michelakis ED, Morrell NW, et al. Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 2009;54:S10–S19. |
| 4. | Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, Christman BW, Weir EK, Eickelberg O, Voelkel NF, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43:13S–24S. |
| 5. | Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, McMurtry IF, Stenmark KR, Thistlethwaite PA, Weissmann N, et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54:S20–S31. |
| 6. | Dewachter L, Adnot S, Fadel E, Humbert M, Maitre B, Barlier-Mur AM, Simonneau G, Hamon M, Naeije R, Eddahibi S. Angiopoietin/tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension. Am J Respir Crit Care Med 2006;174:1025–1033. |
| 7. | Eddahibi S, Guignabert C, Barlier-Mur AM, Dewachter L, Fadel E, Dartevelle P, Humbert M, Simonneau G, Hanoun N, Saurini F, et al. Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: critical role for serotonin-induced smooth muscle hyperplasia. Circulation 2006;113:1857–1864. |
| 8. | Guignabert C, Alvira CM, Alastalo TP, Sawada H, Hansmann G, Zhao M, Wang L, El-Bizri N, Rabinovitch M. Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes pdgf receptor-beta-dependent pulmonary arterial muscularization. Am J Physiol Lung Cell Mol Physiol 2009;297:L1082–L1090. |
| 9. | Izikki M, Guignabert C, Fadel E, Humbert M, Tu L, Zadigue P, Dartevelle P, Simonneau G, Adnot S, Maitre B, et al. Endothelial-derived FGF2 contributes to the progression of pulmonary hypertension in humans and rodents. J Clin Invest 2009;119:512–523. |
| 10. | Sakao S, Tatsumi K, Voelkel NF. Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation. Respir Res 2009;10:95. |
| 11. | Fuks Z, Persaud RS, Alfieri A, McLoughlin M, Ehleiter D, Schwartz JL, Seddon AP, Cordon-Cardo C, Haimovitz-Friedman A. Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo. Cancer Res 1994;54:2582–2590. |
| 12. | Haimovitz-Friedman A, Balaban N, McLoughlin M, Ehleiter D, Michaeli J, Vlodavsky I, Fuks Z. Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis. Cancer Res 1994;54:2591–2597. |
| 13. | Konig A, Menzel T, Lynen S, Wrazel L, Rosen A, Al-Katib A, Raveche E, Gabrilove JL. Basic fibroblast growth factor (bFGF) upregulates the expression of Bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis. Leukemia 1997;11:258–265. |
| 14. | Wieder R, Wang H, Shirke S, Wang Q, Menzel T, Feirt N, Jakubowski AA, Gabrilove JL. Low level expression of basic FGF upregulates Bcl-2 and delays apoptosis, but high intracellular levels are required to induce transformation in NIH 3T3 cells. Growth Factors 1997;15:41–60. |
| 15. | Turner N, Grose R. Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 2010;10:116–129. |
| 16. | Miao HQ, Fritz TA, Esko JD, Zimmermann J, Yayon A, Vlodavsky I. Heparan sulfate primed on beta-d-xylosides restores binding of basic fibroblast growth factor. J Cell Biochem 1995;57:173–184. |
| 17. | Folkman J, Klagsbrun M, Sasse J, Wadzinski M, Ingber D, Vlodavsky I. A heparin-binding angiogenic protein–basic fibroblast growth factor–is stored within basement membrane. Am J Pathol 1988;130:393–400. |
| 18. | Guignabert C, Tu L, Izikki M, Dewachter L, Zadigue P, Humbert M, Adnot S, Fadel E, Eddahibi S. Dichloroacetate treatment partially regresses established pulmonary hypertension in mice with SM22alpha-targeted overexpression of the serotonin transporter. FASEB J 2009;23:4135–4147. |
| 19. | Guignabert C, Raffestin B, Benferhat R, Raoul W, Zadigue P, Rideau D, Hamon M, Adnot S, Eddahibi S. Serotonin transporter inhibition prevents and reverses monocrotaline-induced pulmonary hypertension in rats. Circulation 2005;111:2812–2819. |
| 20. | Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 2002;12:9–18. |
| 21. | Lefevre G, Babchia N, Calipel A, Mouriaux F, Faussat AM, Mrzyk S, Mascarelli F. Activation of the FGF2/FGFr1 autocrine loop for cell proliferation and survival in uveal melanoma cells. Invest Ophthalmol Vis Sci 2009;50:1047–1057. |
| 22. | Masri FA, Xu W, Comhair SA, Asosingh K, Koo M, Vasanji A, Drazba J, Anand-Apte B, Erzurum SC. Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L548–L554. |
| 23. | Ackermann EJ, Taylor JK, Narayana R, Bennett CF. The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem 1999;274:11245–11252. |
| 24. | Fiebig AA, Zhu W, Hollerbach C, Leber B, Andrews DW. Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line. BMC Cancer 2006;6:213. |
| 25. | Simonian PL, Grillot DA, Nunez G. Bcl-2 and Bcl-XL can differentially block chemotherapy-induced cell death. Blood 1997;90:1208–1216. |
| 26. | McMurtry MS, Archer SL, Altieri DC, Bonnet S, Haromy A, Harry G, Bonnet S, Puttagunta L, Michelakis ED. Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension. J Clin Invest 2005;115:1479–1491. |
| 27. | McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K, Michelakis ED. Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis. Circ Res 2004;95:830–840. |
| 28. | Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811–2821. |
| 29. | Letai A, Scorrano L. Laying the foundations of programmed cell death. Cell Death Differ 2006;13:1245–1247. |
| 30. | Bryckaert M, Guillonneau X, Hecquet C, Courtois Y, Mascarelli F. Both FGF1 and Bcl-X synthesis are necessary for the reduction of apoptosis in retinal pigmented epithelial cells by FGF2: role of the extracellular signal-regulated kinase 2. Oncogene 1999;18:7584–7593. |
| 31. | Jost M, Huggett TM, Kari C, Boise LH, Rodeck U. Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-XL expression through a mek-dependent pathway. J Biol Chem 2001;276:6320–6326. |
| 32. | Liu YZ, Boxer LM, Latchman DS. Activation of the Bcl-2 promoter by nerve growth factor is mediated by the P42/P44 MAPK cascade. Nucleic Acids Res 1999;27:2086–2090. |
| 33. | Pugazhenthi S, Miller E, Sable C, Young P, Heidenreich KA, Boxer LM, Reusch JE. Insulin-like growth factor-i induces Bcl-2 promoter through the transcription factor camp-response element-binding protein. J Biol Chem 1999;274:27529–27535. |
| 34. | Song S, Wientjes MG, Gan Y, Au JL. Fibroblast growth factors: an epigenetic mechanism of broad spectrum resistance to anticancer drugs. Proc Natl Acad Sci USA 2000;97:8658–8663. |
| 35. | LeRoith D, Werner H, Beitner-Johnson D, Roberts CT Jr. Molecular and cellular aspects of the insulin-like growth factor i receptor. Endocr Rev 1995;16:143–163. |
| 36. | Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway: requirement for Flk-1/KDR activation. J Biol Chem 1998;273:30336–30343. |
| 37. | Voelkel NF, Cool C, Taraceviene-Stewart L, Geraci MW, Yeager M, Bull T, Kasper M, Tuder RM. Janus face of vascular endothelial growth factor: the obligatory survival factor for lung vascular endothelium controls precapillary artery remodeling in severe pulmonary hypertension. Crit Care Med 2002;30:S251–S256. |
| 38. | Teichert-Kuliszewska K, Kutryk MJ, Kuliszewski MA, Karoubi G, Courtman DW, Zucco L, Granton J, Stewart DJ. Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ Res 2006;98:209–217. |
| 39. | Matsunaga S, Okigaki M, Takeda M, Matsui A, Honsho S, Katsume A, Kishita E, Che J, Kurihara T, Adachi Y, et al. Endothelium-targeted overexpression of constitutively active FGF receptor induces cardioprotection in mice myocardial infarction. J Mol Cell Cardiol 2009;46:663–673. |
| 40. | Hansmann G, de Jesus Perez VA, Alastalo TP, Alvira CM, Guignabert C, Bekker JM, Schellong S, Urashima T, Wang L, Morrell NW, et al. An antiproliferative BMP-2/PPARgamma/apoE axis in human and murine SMCs and its role in pulmonary hypertension. J Clin Invest 2008;118:1846–1857. |
| 41. | Tian J, Smith A, Nechtman J, Podolsky R, Aggarwal S, Snead C, Kumar S, Elgaish M, Oishi P, Goerlach A, et al. Effect of ppargamma inhibition on pulmonary endothelial cell gene expression: gene profiling in pulmonary hypertension. Physiol Genomics 2009;40:48–60. |
