Thrombin Upregulates PAI-1 and Mesothelial–Mesenchymal Transition Through PAR-1 and Contributes to Tuberculous Pleural Fibrosis
Abstract
:1. Introduction
2. Results
2.1. Thrombin Levels Between Transudative Pleural Effusion and Tuberculous Pleural Effusion
2.2. Cytokines and Fibrinolytic Factors between TBPE Patients with Residual Pleural Thickening (RPT) ≤ 10 mm and RPT > 10 mm
2.3. Correlation Between Thrombin and Inflammatory Parameters, Fibrinolytic Factors and Cytokines in TBPE
2.4. Multivariate Logistic Regression Analysis
2.5. Optimal Sensitivity, Specificity, and Cutoff Value of Variables to Predict RPT > 10 mm
2.6. Correlation Between Effusion Thrombin or Plasminogen Activator Inhibitor-1 (PAI-1) and Residual Pleural Fibrosis
2.7. Protease-Activated Receptor (PAR)-1 Silencing Attenuates Pleural Fibrosis in Vivo
2.8. Thrombin Induces Mesothelial–Mesenchymal Transition in Vivo and in Vitro Through PAR-1, PI3K/AKT and NF-κB Signalings
2.9. Thrombin Induces PAI-1 Expression in Human Pleural Mesothelial Cells Through PAR-1/JNK Signaling
2.10. Heat-Killed Mycobacterium Tuberculosis H37Ra Increased Thrombin Production in Human PMCs
2.11. MTBRa Induced mRNA and Protein Expression of Tissue Factor and Prothrombin in Human PMCs
2.12. MTBRa Upregulates PAI-1, α-SMA and Fibronectin Expression Through PAR-1 in Human PMCs
2.13. Expression of PAR-1, Prothrombin, PAI-1, and α-SMA in the Pleural Mesothelium of Patients with TBPE
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Patient Selection
4.3. Thoracentesis and Pleural Fluid Analysis
4.4. Chest Radiographs and Pulmonary Function
4.5. Measurement of Cytokines and Fibrinolytic Factors
4.6. Carbon Black/Bleomycin Pleural Fibrosis Mouse Model
4.7. Cell Cultivation of Pleural Mesothelial Cells
4.8. Protease-Activated Receptor-1 Silencing
4.9. Western Blotting Assay
4.10. PAI-1 Luciferase Activity Assay
4.11. Reverse Transcription–Polymerase Chain Reaction
4.12. Immunofluorescence Staining Assay
4.13. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
α-SMA | α-smooth muscle actin |
CBB | Carbon black/bleomycin |
ECM | Extracelluar matrix |
MMT | Mesothelial–mesenchymal transition |
MTBRa | Mycobacterium tuberculosis H37Ra |
PAI-1 | Plasminogen activator inhibitor-1 |
PAR-1 | Protease-activated receptor-1 |
PMC | Pleural mesothelial cell |
RPT | Residual pleural thickening |
TBPE | Tuberculous pleural effusion |
TF | Tissue factor |
TPE | Transudative pleural effusion |
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TPE | TBPE | p-Value * | ||
---|---|---|---|---|
RPT ≤ 10 mm | RPT > 10 mm | |||
Subject, n | 22 | 14 | 10 | |
Age, years | 80 (65–88) | 64 (20–87) | 79 (34–91) | 0.186 |
Males, n | 14 | 9 | 6 | 0.830 |
Symptom onset to enrollment, days | 7 (5–10) | 14 (6–20) | 16 (7–25) | 0.304 |
Pleural fluid | ||||
pH value | 7.39 (7.30–7.46) | 7.37 (7.25–7.46) | 7.30 (7.23–7.37) | 0.010 |
Glucose, mg/dL | 141 (102–193) | 126 (68–175) | 88 (25–181) | 0.105 |
Protein, g/L | 2.3 (1.1–3.6) | 4.7 (3.3–5.8) | 4.6 (3.6–6.5) | 0.829 |
LDH, IU/dL | 83 (22–153) | 274 (121–1370) | 495 (215–2001) | 0.064 |
Leukocyte count, cells/μL | 313 (108–1080) | 959 (235–5840) | 1531 (137–6872) | 0.667 |
ADA, IU/L | 13 (6–36) | 112 (59–262) | 180 (68–220) | 0.045 |
Thrombin, pg/mL | 0.7 (0.1–1.1) | 5.0 (3.9–5.4) | 5.7 (5.4–6.7) | <0.0001 |
PAI-1, ng/mL | 36.6 (3.1–140.5) | 107.2 (40.4–150.1) | 137.5 (100.9–225.3) | 0.003 |
tPA, ng/mL | 12.1 (6.9–35.0) | 24.1 (1.0–56.9) | 14.1 (1.8–41.5) | 0.186 |
TNF-α, pg/mL | 17.0 (4.5–109.8) | 37.7 (15.0–184.4) | 88.8 (35.8–287.2) | 0.025 |
IL-1β, pg/mL | 1.7 (1.4–3.7) | 5.9 (4.3–34.3) | 17.6 (5.8–63.1) | 0.021 |
Initial pleural effusion CXR score, % | 49 (25–65) | 43 (29–59) | 61 (42–93) | 0.003 |
Residual pleural shadowing CXR score, % | 0 (0–1) | 2.0 (0.6–4.0) | 4.5 (2.5–5.9) | <0.001 |
FVC at 12 months, % predicted | 79 (77–82) | 80 (78–81) | 74 (71–76) | <0.001 |
Variables | Coefficient * | p-Value |
---|---|---|
pH value | −0.46 | 0.051 |
Glucose, mg/dL | −0.16 | 0.460 |
LDH, IU/dL | 0.35 | 0.070 |
ADA, IU/L | 0.40 | 0.063 |
PAI-1, ng/mL | 0.65 | <0.0001 |
tPA, ng/mL | −0.04 | 0.862 |
TNF-α, pg/mL | 0.38 | 0.066 |
IL-1β, pg/mL | 0.41 | 0.058 |
Variables | Thrombin Excluded | PAI-1 Excluded | ||||
---|---|---|---|---|---|---|
OR | 95% CI | p-Value | OR | 95% CI | p-Value | |
Thrombin, U/mL | 9.05 | 3.09–28.42 | 0.007 | |||
pH value | 0.92 | 0.83–1.02 | 0.135 | 0.25 | 0.01–1.181 | 0.623 |
ADA, IU/L | 1.29 | 0.77–1.83 | 0.609 | 1.12 | 0.99–1.26 | 0.721 |
TNF-α, pg/mL | 1.33 | 0.91–1.95 | 0.147 | 1.04 | 0.95–1.09 | 0.925 |
IL-1β, pg/mL | 1.02 | 0.98–1.07 | 0.272 | 1.08 | 0.95–1.18 | 0.182 |
PAI-1, ng/mL | 2.26 | 1.05–4.87 | 0.037 |
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Hsieh, C.-Y.; Sheu, J.-R.; Yang, C.-H.; Chen, W.-L.; Tsai, J.-H.; Chung, C.-L. Thrombin Upregulates PAI-1 and Mesothelial–Mesenchymal Transition Through PAR-1 and Contributes to Tuberculous Pleural Fibrosis. Int. J. Mol. Sci. 2019, 20, 5076. https://doi.org/10.3390/ijms20205076
Hsieh C-Y, Sheu J-R, Yang C-H, Chen W-L, Tsai J-H, Chung C-L. Thrombin Upregulates PAI-1 and Mesothelial–Mesenchymal Transition Through PAR-1 and Contributes to Tuberculous Pleural Fibrosis. International Journal of Molecular Sciences. 2019; 20(20):5076. https://doi.org/10.3390/ijms20205076
Chicago/Turabian StyleHsieh, Cheng-Ying, Joen-Rong Sheu, Chih-Hao Yang, Wei-Lin Chen, Jie-Heng Tsai, and Chi-Li Chung. 2019. "Thrombin Upregulates PAI-1 and Mesothelial–Mesenchymal Transition Through PAR-1 and Contributes to Tuberculous Pleural Fibrosis" International Journal of Molecular Sciences 20, no. 20: 5076. https://doi.org/10.3390/ijms20205076