Endothelin-1 Induces Mesothelial Mesenchymal Transition and Correlates with Pleural Fibrosis in Tuberculous Pleural Effusions

Endothelin (ET)-1 is involved in various fibrotic diseases. However, its implication in pleural fibrosis remains unknown. We aimed to study the profibrotic role of ET-1 in tuberculous pleural effusion (TBPE). The pleural effusion ET-1 levels were measured among 68 patients including transudative pleural effusion (TPE, n = 12), parapneumonic pleural effusion (PPE, n = 20), and TBPE (n = 36) groups. Pleural fibrosis, defined as radiological residual pleural thickening (RPT) and shadowing, was measured at 12-month follow-up. Additionally, the effect of ET-1 on mesothelial mesenchymal transition (MMT) and extracellular matrix (ECM) producion in human pleural mesothelial cells (PMCs) was assessed. Our findings revealed that effusion ET-1 levels were significantly higher in TBPE than in TPE and PPE, and were markedly higher in TBPE patients with RPT >10 mm than those with RPT ≤10 mm. ET-1 levels correlated substantially with residual pleural shadowing and independently predicted RPT >10 mm in TBPE. In PMCs, ET-1 time-dependently induced MMT with upregulation of α-smooth muscle actin and downregulation of E-cadherin, and stimulated ECM production; furthermore, ET receptor antagonists effectively abrogated these effects. In conclusion, ET-1 induces MMT and ECM synthesis in human PMCs and correlates with pleural fibrosis in TBPE. This study confers a novel insight into the pathogenesis and potential therapies for fibrotic pleural diseases.


Introduction
Endothelin (ET)-1, initially identified as a potent vasoconstrictor, has been substantially implicated in the pathogenesis of inflammation and fibrosis of various organs [1]. In the context of lung diseases, plasma ET-1 levels were elevated in patients with acute respiratory distress syndrome [2], and endothelin blockers reduced lipopolysaccharide-induced lung inflammation in vivo [3]. Additionally, increased expression of ET-1 was observed in lung tissues from patients with idiopathic pulmonary

Human Pleural Mesothelial Cell (PMC) Culture
The primary human PMCs were harvested from pleural fluids of patients with congestive heart failure and cultured as previously described [19]. The Met-5A human pleural mesothelial cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in medium 199 (GIBCO TM , Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO 2 at 37 • C as described previously [19].

Western Blot
Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting membranes were incubated with a specific antibody against ET-1, α-SMA, E-cadherin, collagen I and fibronectin, AKT, IκBα and α-tubulin. The quantitative densitometric analysis was conducted as formerly reported [19].

Phase Contrast Microscopy
The cultured primary PMCs were left untreated (control) or treated with ET-1 for various indicated times (24,48,72, and 96 h), and the phenotypic changes were determined by phase contrast microscopy [13].

Immunofluorescence Staining
Primary or MeT-5A human PMCs were cultured on collagen (Vitrogen ® )-coated glass cover slips for 24 h and change to FBS-free medium for another 24 h. Then, for the MMT phenotype study, primary PMCs were treated with or without ET-1 (10 nM) for 72 h and then incubated with primary antibodies: mouse anti-E-cadherin and mouse anti-α-SMA and respective secondary antibody conjugated with fluorescein isothiocyanate (FITC), and viewed by immunofluorescence microscopy.
For evaluation of the effect of ET receptor antagonists on ET-1-induced α-SMA expression, MeT-5A cells were pretreated with PBS, BQ123, BQ788 or Bosentan for 15 min, then treated with ET-1 (10 nM) for 24 h, and incubated with primary antibody to α-SMA. After three washings with PBS, the cells were incubated with secondary FITC-conjugated antibody for 1 h at room temperature.
For measurement of ET-1 expression and MMT in pleural mesothelium, the paraffin-embedded parietal pleura tissue biopsied from patients with TBPE were placed onto glass slides, de-waxed in xylene and alcohols, washed twice with PBS, and then immunostained with primary antibodies to mesothelin, ET-1 or α-SMA, followed by FITC conjugated secondary antibody. Cells were also co-stained with 4', 6-diamidino-2-phenylindole (DAPI) to visualize nuclei. The stained cells were mounted with anti-fading mounting medium and scanned on the TCS SP5 confocal spectral microscope imaging system (Leica, Wetzlar, Germany) as previously described [21].

Statistical Analysis
Quantitative data are presented as median (range) or mean ± SD. Comparisons of continuous data were made using the Kruskal-Wallis test or one way analysis of variance (ANOVA) among three groups, and Mann-Whitney U test or unpaired t-test between two groups, where appropriate. The correlations between variables were determined by Spearman rank correlation coefficients. Categorical variables between two groups were examined using the χ 2 method and/or Fisher's exact test, when appropriate.
Multivariate logistic regression analyses were performed to determine factors independently associated with development of RPT >10 mm. Variables found to be significant in the univariate analysis were entered into a binary logistic regression analysis. Results of multivariable analyses are reported as odds ratios with 95% confidence intervals and p-values. The optimal sensitivity, specificity and cutoff value of pleural fluid variables to predict RPT >10 mm were evaluated with the receiver operating characteristics (ROC) by analyzing the area under the curve. A two-tailed p-value < 0.05 was considered to be statistically significant.

Endothelin (ET)-1 Levels among Transudative Pleural Effusion (TPE), Parapneumonic Pleural Effusion (PPE) and Tuberculous Pleural Effusion (TBPE) Groups
Consecutive 68 patients with TPE (n = 12), PPE (n = 20) and TBPE (n = 36) were enrolled (Table 1), including 42 men and 26 women with an age range from 20 to 95 years. All patients with TPE were diagnosed with congestive heart failure, and the PPE group consisted of 8 uncomplicated PPE and 12 complicated PPE. All patients finished 12 months of follow-up from January 2011 through December 2014.

Cytokines and Fibrinolytic Factors between TBPE Patients with RPT ≤10 mm and RPT >10 mm
In pleural fluid analysis, the levels of pH, glucose and LDH reflect the degree of inflammation [22], the balance of PAI-1 and tPA determines the fibrinolytic activity [11], the proinflammatory cytokines TNF-α and IL-1β accentuate inflammatory responses [23], and the profibrotic cytokine TGF-β1 mediates fibrosis in the pleural space [12]. The effusion ADA level is useful for diagnosis of TBPE [10], although its relationship with pleural fibrosis remains unknown. Therefore, we further compared these pleural fluid characteristics, ADA, TNF-α, IL-1β, TGF-β1 and fibrinolytic factors PAI-1 and tPA between the two groups ( Table 2). The pleural fluid parameters demonstrated that RPT >10 mm group had significantly lower levels of effusion pH and ADA than did RPT ≤10 mm group, while there was no considerable difference in pleural fluid values of glucose, LDH, and leukocyte count between two groups. Moreover, besides ET-1, the effusion levels of PAI-1, TNF-α, IL-1β, and especially TGF-β1 were significantly higher in RPT >10 mm group than in RPT ≤10 mm group. Additionally, the former had greater initial effusion CXR score on presentation than the latter. Furthermore, patients with RPT >10 mm had significant lower forced vital capacity than did those with RPT ≤10 mm at 12 months. These findings imply that the increased inflammation, decreased fibrinolysis and elevated profibrotic cytokines, including ET-1 and TGF-β1, may be associated with the development of pleural fibrosis in TBPE.

Cytokines and Fibrinolytic Factors between TBPE Patients with RPT ≤10 mm and RPT >10 mm
In pleural fluid analysis, the levels of pH, glucose and LDH reflect the degree of inflammation [22], the balance of PAI-1 and tPA determines the fibrinolytic activity [11], the proinflammatory cytokines TNF-α and IL-1β accentuate inflammatory responses [23], and the profibrotic cytokine TGF-β1 mediates fibrosis in the pleural space [12]. The effusion ADA level is useful for diagnosis of TBPE [10], although its relationship with pleural fibrosis remains unknown. Therefore, we further compared these pleural fluid characteristics, ADA, TNF-α, IL-1β, TGF-β1 and fibrinolytic factors PAI-1 and tPA between the two groups ( Table 2). The pleural fluid parameters demonstrated that RPT >10 mm group had significantly lower levels of effusion pH and ADA than did RPT ≤10 mm group, while there was no considerable difference in pleural fluid values of glucose, LDH, and leukocyte count between two groups. Moreover, besides ET-1, the effusion levels of PAI-1, TNF-α, IL-1β, and especially TGF-β1 were significantly higher in RPT >10 mm group than in RPT ≤10 mm group. Additionally, the former had greater initial effusion CXR score on presentation than the latter. Furthermore, patients with RPT >10 mm had significant lower forced vital capacity than did those with RPT ≤10 mm at 12 months. These findings imply that the increased inflammation, decreased fibrinolysis and elevated profibrotic cytokines, including ET-1 and TGF-β1, may be associated with the development of pleural fibrosis in TBPE.

Correlation between ET-1 and Inflammatory Parameters, Fibrinolytic Factors and Cytokines in TBPE
Accordingly, to explore the link between ET-1 and inflammation, fibrinolysis and fibrosis in TBPE, we examined the relationship between ET-1 and inflammatory parameters, fibrinolytic factors and other cytokines among TBPE patients ( Table 3). The results demonstrated that the effusion levels of ET-1 were positively correlated with those of TGF-β1 (r = 0.45, p = 0.008), and negatively correlated with those of glucose (r = −0.35, p = 0.036). However, there was no significant correlation between ET-1 and pH, LDH, PAI-1, tPA, TNF-α and IL-1β, respectively. The current data suggest that ET-1 may have greater association with fibrosis than with inflammation or fibrinolysis in TBPE.

Correlation between Effusion ET-1, TGF-β1 and Residual Pleural Fibrosis
Given the predictive value of effusion ET-1 and TGF-β1, the correlation between ET-1, TGF-β1 and residual pleural fibrosis were analyzed. As shown in Figure 2C,D, the effusion levels of ET-1 were positively correlated with the CXR score of pleural shadowing at 12 months; however, there was no significant correlation between TGF-β1 and residual pleural shadowing, suggesting that ET-1 is substantially implicated in fibrogenesis of TBPE that merits further experiments to confirm.

Heat-Killed Mycobacterium tuberculosis H37Ra (MTBRa) Induces ET-1 Production in Human PMCs
To elucidate the marked increase of ET-1 in TBPE ( Figure 1A), we studied the ET-1 production by primary human PMCs upon MTBRa stimulation. As shown in Figure 3A, ET-1 expression increased significantly upon stimulation with various concentrations of MTBRa (0.1, 1, 10 and 100 ng/mL) for 24 h. Consistently, the ET-1 level in the supernatants of PMC culture stimulated with MTBRa for 24 h increased in a concentration-dependent manner as compared with the control ( Figure 3B). These

Heat-Killed Mycobacterium tuberculosis H37Ra (MTBRa) Induces ET-1 Production in Human PMCs
To elucidate the marked increase of ET-1 in TBPE ( Figure 1A), we studied the ET-1 production by primary human PMCs upon MTBRa stimulation. As shown in Figure 3A, ET-1 expression increased significantly upon stimulation with various concentrations of MTBRa (0.1, 1, 10 and 100 ng/mL) for 24 h. Consistently, the ET-1 level in the supernatants of PMC culture stimulated with MTBRa for 24 h increased in a concentration-dependent manner as compared with the control ( Figure  3B). These findings suggest that, upon Mycobacterium tuberculosis (M. tuberculosis) infection, human PMCs are activated to produce a considerable amount of ET-1 that contribute to the elevated levels in TBPE.

ET-1 Induces MMT and ECM Synthesis in PMCs
Subsequently, to verify the profibrotic effect of ET-1 on pleural mesothelium, the human PMCs were treated with or without 10 nM of ET-1 for 96 h. The primary human PMCs in the absence of ET-1 (control) showed typical cobblestone appearance, whereas ET-1-treated PMCs revealed substantial phenotypic change after 24 h and transformed into fibroblast-like appearance with elongated, spindle-shaped morphology ( Figure 4A). Consistently, ET-1 significantly increased α-SMA while decreased E-cadherin expression in PMCs, compared to the resting condition ( Figure 4B). Additionally, to further validate that the MMT will lead to ECM synthesis, we also examined the expression of collagen I and fibronectin in PMCs activated with ET-1. A significant increase in the production of collagen I and fibronectin was noted after 24 h treatment of ET-1 in a time-dependent manner, with maximal response occurring at 72 h ( Figure 4B). Similarly, the immunofluorescence staining for E-cadherin and α-SMA revealed MMT phenotypic change and downregulation of E-cadherin and upregulation of α-SMA in ET-1-treated PMCs compared with the resting controls ( Figure 4C). These findings indicated that ET-1 stimulates human PMCs to undergo MMT and produce ECM and thereby contributes to pleural fibrosis.

ET-1 Induces MMT and ECM Synthesis in PMCs
Subsequently, to verify the profibrotic effect of ET-1 on pleural mesothelium, the human PMCs were treated with or without 10 nM of ET-1 for 96 h. The primary human PMCs in the absence of ET-1 (control) showed typical cobblestone appearance, whereas ET-1-treated PMCs revealed substantial phenotypic change after 24 h and transformed into fibroblast-like appearance with elongated, spindle-shaped morphology ( Figure 4A). Consistently, ET-1 significantly increased α-SMA while decreased E-cadherin expression in PMCs, compared to the resting condition ( Figure 4B). Additionally, to further validate that the MMT will lead to ECM synthesis, we also examined the expression of collagen I and fibronectin in PMCs activated with ET-1. A significant increase in the production of collagen I and fibronectin was noted after 24 h treatment of ET-1 in a time-dependent manner, with maximal response occurring at 72 h ( Figure 4B). Similarly, the immunofluorescence staining for E-cadherin and α-SMA revealed MMT phenotypic change and downregulation of Ecadherin and upregulation of α-SMA in ET-1-treated PMCs compared with the resting controls ( Figure 4C). These findings indicated that ET-1 stimulates human PMCs to undergo MMT and produce ECM and thereby contributes to pleural fibrosis.

Expression of ET-1 and α-SMA in the Pleural Mesothelium of TBPE
Furthermore, the immunofluorescence staining was performed on paraffin-embedded pleural biopsy specimens from patients with TBPE ( Figure 4D), and demonstrated proliferation of PMCs (mesothelin positive cells) into multilayers that exhibited substantial dual expression of ET-1 + /mesothelin + and α-SMA + /mesothelin + , which further indicates the important implication of ET-1 and MMT in the pathogenesis of TBPE.

MTBRa-Induced Mesothelial Mesenchymal Transition (MMT) and Extracellular Matrix (ECM) Synthesis in PMCs via ET-1
Accordingly, MTBRa was used to stimulate PMCs to investigate the role of ET-1 and MMT in TBPE. It demonstrated that MTBRa concentration-dependently reduced E-cadherin expression, induced α-SMA synthesis, and elicited production of collagen I and fibronectin in MeT-5A cells ( Figure 5A). Conversely, pretreatment with the ET-A receptor antagonist (BQ-123), ET-B receptor antagonist (BQ-788) and dual ET receptor antagonist (Bosentan) all abrogated the decrease in Ecadherin and increase in α-SMA, collagen I and fibronectin caused by MTBRa ( Figure 5B). Together, (B) Primary human PMCs were treated with ET-1 (10 nM) for the indicated times, and E-cadherin, α-SMA, collagen I and fibronectin were analyzed by Western blotting. Results are representative of three independent experiments. (C) Primary human PMCs were treated with or without (control) ET-1 for 72 h, and phenotypic change and expression for E-cadherin and α-SMA were evaluated by immunofluorescence microscopy. Scale bar, 20 µm. (D) Immunofluorescence staining for ET-1 (red) or α-SMA (red) and mesothelin (green) in PMCs was performed on paraffin-embedded pleural biopsy specimens from patients with TBPE and observed by confocal microscopy. Cells were also co-stained with DAPI to visualize the nuclei (blue). Scale bar, 50 µm. MMT, mesothelial mesenchymal transition; ECM, extracellular matrix; PMC, pleural mesothelial cell; α-SMA, α-smooth muscle actin; DAPI, 4', 6-diamidino-2-phenylindole.

Expression of ET-1 and α-SMA in the Pleural Mesothelium of TBPE
Furthermore, the immunofluorescence staining was performed on paraffin-embedded pleural biopsy specimens from patients with TBPE ( Figure 4D), and demonstrated proliferation of PMCs (mesothelin positive cells) into multilayers that exhibited substantial dual expression of ET-1 + /mesothelin + and α-SMA + /mesothelin + , which further indicates the important implication of ET-1 and MMT in the pathogenesis of TBPE.

MTBRa-Induced Mesothelial Mesenchymal Transition (MMT) and Extracellular Matrix (ECM) Synthesis in PMCs via ET-1
Accordingly, MTBRa was used to stimulate PMCs to investigate the role of ET-1 and MMT in TBPE. It demonstrated that MTBRa concentration-dependently reduced E-cadherin expression, induced α-SMA synthesis, and elicited production of collagen I and fibronectin in MeT-5A cells ( Figure 5A). Conversely, pretreatment with the ET-A receptor antagonist (BQ-123), ET-B receptor antagonist (BQ-788) and dual ET receptor antagonist (Bosentan) all abrogated the decrease in E-cadherin and increase in α-SMA, collagen I and fibronectin caused by MTBRa ( Figure 5B). Together, these findings indicate that MTBRa stimulates MMT and ECM production in PMCs via the action of ET-1.

ET-1 Induces MMT in PMCs Mainly through Activation of ET-A Receptor
Furthermore, we explored which ET receptor subtype mediates the action of ET-1 on MMT in PMCs. The data showed that pretreatment with BQ-123 or Bosentan significantly attenuated ET-1−induced downregulation of E-cadherin and upregulation of α-SMA, while BQ-788 had the same effect but to a lesser degree ( Figure 5C). In parallel, the immunofluorescence study revealed that both BQ-123 and Bosentan, while BQ-788 less prominently, inhibited the ET-1−stimulated increase in α-SMA expression ( Figure 5D). Moreover, treatment with three ET receptor antagonists all substantially ablated the expression of ECM proteins ( Figure 5C). These results indicate that ET-1 induces MMT predominantly through activation of ET-A receptor.

ET-1−Induced α-SMA Expression is PI3K/AKT and NF-κB Dependent
To further investigate the mechanism underlying the ET-1−activated MMT in PMCs, we proceeded to identify the involved cellular signaling. The data demonstrated ET-1 significantly induced α-SMA in MeT-5A cells in a concentration-dependent manner with a maximal response occurring with 10 nM ( Figure 6A). Besides, to recognize whether the increase in α-SMA protein expression after ET-1 stimulation resulted from the induction of α-SMA mRNA expression, RT-PCR analysis was performed. After treatment with ET-1 for 24, 48, and 72 hours, respectively, MeT-5A cells had significant increase in α-SMA mRNA expression at the concentration of 10 nM ET-1 ( Figure 6B). Furthermore, as shown in Figure 6C, pretreatment with neither an MEK inhibitor (PD98059; 20 µM), a JNK inhibitor (SP600125; 10 µM) nor a p38 MAPK inhibitor (SB203580; 10 µM) attenuated α-SMA expression induced by ET-1. In contrast, a NF-κB inhibitor (Parthenolide; 10 µM) and a PI3K inhibitor (LY294002; 20 µM) significantly reduced ET-1−stimulated α-SMA protein production. Consistently, ET-1 significantly induced AKT phosphorylation ( Figure 6D) and inhibitor of κB-α (IκB-α) degradation ( Figure 6E) in a time-dependent manner. Collectively, these findings demonstrated that ET-1 induced MMT in PMCs is PI3K/AKT and NF-κB dependent and possibly through activation of ET-A receptor and PI3K/AKT/NF-κB signaling (Figure 7). 1−induced downregulation of E-cadherin and upregulation of α-SMA, while BQ-788 had the same effect but to a lesser degree ( Figure 5C). In parallel, the immunofluorescence study revealed that both BQ-123 and Bosentan, while BQ-788 less prominently, inhibited the ET-1−stimulated increase in α-SMA expression ( Figure 5D). Moreover, treatment with three ET receptor antagonists all substantially ablated the expression of ECM proteins ( Figure 5C). These results indicate that ET-1 induces MMT predominantly through activation of ET-A receptor.

Discussion
The present study demonstrated that the levels of effusion ET-1 were significantly higher in TBPE than in TPE and PPE, and markedly greater in TBPE patients with RPT >10 mm than those with RPT ≤10 mm. Moreover, ET-1 levels correlated significantly with residual pleural shadowing of TBPE and could predict pleural fibrosis at the end of 12-month follow-up. The in vitro experiments showed that MTBRa significantly stimulated human PMCs to produce ET-1 which time-dependently induced MMT with downregulation of E-cadherin and upregulation of α-SMA, collagen I and fibronectin in human PMCs, whereas pretreatment with ET-A receptor antagonist substantially attenuated all the effects. Moreover, concomitant expression of ET-1 and α-SMA was demonstrated in the pleural mesothelium of patients with TBPE, and ET-1 significantly increased α-SMA expression through triggering AKT phosphorylation and NF-κB activation. To our knowledge, this is the first study to signify the expression and profibrotic implication of ET-1 and MMT in TBPE and to demonstrate the regulatory mechanism underlying the ET-1−induced MMT and ECM production in human PMCs.
ET-1 has been associated with the pathogenesis of numerous infectious diseases [24], though its role in pleural infections remains elusive. Besides, the link between ET-1 and M. tuberculosis infection has rarely been explored. A previous in vivo study showed that the blockade of ET-1 activity by

Discussion
The present study demonstrated that the levels of effusion ET-1 were significantly higher in TBPE than in TPE and PPE, and markedly greater in TBPE patients with RPT >10 mm than those with RPT ≤10 mm. Moreover, ET-1 levels correlated significantly with residual pleural shadowing of TBPE and could predict pleural fibrosis at the end of 12-month follow-up. The in vitro experiments showed that MTBRa significantly stimulated human PMCs to produce ET-1 which time-dependently induced MMT with downregulation of E-cadherin and upregulation of α-SMA, collagen I and fibronectin in human PMCs, whereas pretreatment with ET-A receptor antagonist substantially attenuated all the effects. Moreover, concomitant expression of ET-1 and α-SMA was demonstrated in the pleural mesothelium of patients with TBPE, and ET-1 significantly increased α-SMA expression through triggering AKT phosphorylation and NF-κB activation. To our knowledge, this is the first study to signify the expression and profibrotic implication of ET-1 and MMT in TBPE and to demonstrate the regulatory mechanism underlying the ET-1−induced MMT and ECM production in human PMCs.

Discussion
The present study demonstrated that the levels of effusion ET-1 were significantly higher in TBPE than in TPE and PPE, and markedly greater in TBPE patients with RPT >10 mm than those with RPT ≤10 mm. Moreover, ET-1 levels correlated significantly with residual pleural shadowing of TBPE and could predict pleural fibrosis at the end of 12-month follow-up. The in vitro experiments showed that MTBRa significantly stimulated human PMCs to produce ET-1 which time-dependently induced MMT with downregulation of E-cadherin and upregulation of α-SMA, collagen I and fibronectin in human PMCs, whereas pretreatment with ET-A receptor antagonist substantially attenuated all the effects. Moreover, concomitant expression of ET-1 and α-SMA was demonstrated in the pleural mesothelium of patients with TBPE, and ET-1 significantly increased α-SMA expression through triggering AKT phosphorylation and NF-κB activation. To our knowledge, this is the first study to signify the expression and profibrotic implication of ET-1 and MMT in TBPE and to demonstrate the regulatory mechanism underlying the ET-1−induced MMT and ECM production in human PMCs.
ET-1 has been associated with the pathogenesis of numerous infectious diseases [24], though its role in pleural infections remains elusive. Besides, the link between ET-1 and M. tuberculosis infection has rarely been explored. A previous in vivo study showed that the blockade of ET-1 activity by antagonism might lead to progression of tuberculous lung lesions, indicating that ET pathways play a regulatory role in M. tuberculosis infection [25]. We first demonstrated that ET-1 level was markedly higher in TBPE than in TPE, while not significantly elevated in PPE, and that MTBRa induced substantial production of ET-1 in human PMCs, suggesting the distinctive pathogenic role of ET-1 in TBPE.
To ascertain the involvement of ET-1 in the inflammation, fibrinolysis and fibrosis in TBPE, correlation analysis was conducted and showed that ET-1 correlated positively with the most potent profibrotic cytokine TGF-β1. However, no significant correlation existed between ET-1 and inflammatory parameters, fibrinolytic factors and proinflammatory cytokines, respectively. Accordingly, to further clarify the implication of ET-1 in pleural fibrosis, we measured the residual pleural opacity of all TBPE patients and found that both effusion ET-1 and TGF-β1 were remarkably higher in patients with RPT >10 mm than those with RPT ≤10 mm. Moreover, ET-1, compared to TGF-β1, was a better predictor for RPT >10 mm and had stronger positive correlation with the area of residual pleural opacity of TBPE. Nonetheless, as studies have demonstrated the mutual regulation and collaboration between TGF-β1 and ET-1 in the development of pulmonary fibrosis [26], the current data does not imply the superiority of ET-1 over TGF-β1 in the modulation of pleural fibrogenesis, but features the importance of ET-1 in TBPE. Taken together, these findings suggest that ET-1, although not linked to increased inflammation or disordered fibrinolysis, is essential for tuberculous pleural fibrosis, probably acting via induction of aberrant cellular response to injury such as cellular mesenchymal transition and ECM overproduction [5][6][7].
Increasing evidence demonstrated that PMCs experience mesenchymal transition upon stimulation by TGF-β1 or bleomycin [12][13][14][15]. In parallel, we demonstrated that ET-1 time−dependently induced protein expression of the myofibroblast−specific marker, α-SMA, while reduced the epithelial cell marker, E-cadherin, and led to phenotypic alteration in primary human PMCs. Concomitantly, ET-1 stimulated human PMCs to produce collagen I and fibronectin that further verify the fibrogenic activity of PMCs in response to ET-1. Moreover, we demonstrated the concurrent expression of ET-1 and α-SMA in the pleural mesothelium of patients with TBPE and that MTBRa stimulated MMT and ECM synthesis in PMCs through the actions of ET-1. Collectively, these results indicate that ET-1 is an important mediator for MMT in PMCs and may, thereby, contribute to pleural fibrosis in TBPE.
We further investigated the involved receptors and signalings mediating the ET-1−induced MMT and showed that treatment with ET-A receptor antagonist (BQ-123) effectively reversed the loss of E-cadherin and gain of α-SMA in PMCs stimulated by ET-1, while ET-B receptor antagonist (BQ-788) had a similar but lesser effect. This may be due to either lower affinity for or diminished amount of the ET-B receptor on PMCs. Nonetheless, both ET-A and ET-B blockers efficiently repressed the production of collagen I and fibronectin, which may suggest the distinct regulation between MMT and ECM synthesis in PMCs upon ET-1 activation. Furthermore, ET-1 activated PI3K/AKT and NF-κB signaling and blockade of PI3K/AKT and NF-κB significantly attenuated ET-1-upregulated α-SMA expression in PMCs. Altogether, these findings, in line with previous reports [5,6,15], indicated that ET-1 induces MMT in PMC mainly through activation of ET-A receptor and is PI3K/AKT and NF-κB dependent.
As for the therapeutic implication of ET-1 for pleural fibrosis, previous reports have demonstrated that ET receptor antagonist treatment efficiently attenuated ET-1-mediated alveolar EMT in vitro [5,6], mitigated bleomycin-induced lung fibrosis in vivo [27], and displayed a favorable trend to improve progression free survival of histology-proven IPF patients [28,29]. Additionally, recent in vivo studies have employed the heme oxygenase-1 inducer, carbon monoxide or glycogen synthase kinase-3β inhibitor, to attenuate MMT in PMCs that significantly abrogated pleural fibrosis [30,31]. In parallel, our data signified the antifibrotic potential of ET receptor antagonism by concurrently reversing MMT and reducing ECM production in human PMCs. However, the pleural biopsy specimens from patients at diagnosis of TBPE revealed that the MMT of pleural mesothelium occurred early in the disease process ( Figure 4D), implying that prevention of MMT may not be feasible. Nevertheless, the reversing effect of ET blockers on MMT ( Figure 5B-D) may infer a therapeutic potential. Accordingly, more in vivo and clinical studies to inhibit ET-1 as well as MMT are needed for developing novel agents for treatment of pleural fibrosis. Collectively, the current study highlights the role of ET-1 and MMT in the fibrogenesis of TBPE and warrants further investigations of targeting ET-1 and/or MMT as treatment strategies for pleural fibrosis.

Conclusions
In conclusion, ET-1 induces MMT and ECM synthesis in human PMCs and correlates with pleural fibrosis in TBPE. This study confers a novel insight into the pathogenesis and potential therapies for fibrotic pleural diseases.