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Repeated Thoracenteses Affect Proinflammatory Cytokines, Vascular Endothelial Growth Factor, and Fibrinolytic Activity in Pleural Transudates
Repeated Thoracenteses Affect Proinflammatory Cytokines, Vascular Endothelial Growth Factor, and Fibrinolytic Activity in Pleural Transudates CHI-LI CHUNG, MD, MS; CHING-YU YEH, MD; JOEN-RONG SHEU, PHD; YI-CHU CHEN, BS; SHI-CHUAN CHANG, MD, PHD
ABSTRACT: Background: Repeated thoracenteses is indicated in patients with refractory, symptomatic transudative effusions. However, their effect on cytokines and fibrinolytic activity in pleural transudates remains unclear. Methods: Twenty-one patients with symptomatic, large amount of free-flowing transudative effusions caused by heart failure were studied. Thoracentesis with drainage of 500 mL of pleural fluid per day was done for 3 consecutive days (days 1 to 3). Pleural fluid characteristics, tumor necrosis factor (TNF)-␣, interleukin (IL)1, IL-8, vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured during each tap. Chest ultrasonography was done on day 6 to detect the fibrin strands in pleural effusion and the outcome of effusion was evaluated within 7 days after repeated thoracenteses. Results: Effusion levels of lactate dehydrogenase, neutrophils, TNF-␣, IL-1, IL-8,
VEGF, and PAI-1 increased significantly during repeated thoracenteses. Furthermore, the values of PAI-1 and PAI-1/tPA obtained on days 2 and 3 were highly correlated with those of TNF-␣, IL-1, IL-8, and VEGF. On day 6, pleural fibrins were observed on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of TNF-␣, IL-1, VEGF, and PAI-1 on day 2 and day 3, and recurrence of symptomatic effusion after repeated thoracenteses were significantly higher in fibrinous group. Conclusions: Repeated thoracenteses may induce local release of proinflammatory cytokines, VEGF and PAI-1, which may result in fibrin deposition and impair resolution of pleural transudates. KEY INDEXING TERMS: Fibrinogenesis; Pleural effusion; Proinflammatory cytokine; Thoracentesis; Transudates. [Am J Med Sci 2007;334(6):452–457.]
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transudative effusion, which in general shows poor response to medical treatment.1 Occasionally, transudative pleural effusions may reaccumulate rapidly in patients with refractory heart failure and require repeated thoracenteses.1 Fibrinogenesis, defined as fibrin formation in the pleural cavity, induced by repeated thoracenteses, is not uncommon in pleural exudates as detected by chest ultrasonography.3,4 However, to the best of our knowledge, it remains unknown whether repeated thoracenteses can induce fibrinogenesis in transudative effusions, in which the pleura is considered to be intact and noninflammatory. Fibrin turnover in the pleural cavity is greatly affected by the activity of fibrinolysis. The formation of key enzyme in fibrinolysis, plasmin, is based on the equilibrium between plasminogen activators (PAs) and plasminogen activator inhibitors (PAIs).5 Fibrin formation and deposition are the hallmarks of pleural inflammation,6 which may enhance the release of proinflammatory cytokines such as tumor
ransudative pleural effusion occurs when hydrostatic and oncotic pressures across the pleural membrane are altered.1 Congestive heart failure is the most common cause of transudative pleural effusions, which may aggravate the dyspnea in these patients.2 Therapeutic thoracentesis may be indicated, particularly in those with a large amount of
From the Department of Chest Medicine, Taipei Medical University Hospital (CLC, CYY); Graduate Institute of Clinical Medicine (CLC); Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University (JRS); Chest Department, Taipei Veterans General Hospital (YCC, SCC); and Institute of Emergency and Critical Care Medicine (SCC), School of Medicine, National Yang-Ming University, Taipei, Taiwan. Submitted March 15, 2007; accepted in revised form May 22, 2007. This work was supported by grants from the Taipei Medical University (TMU92-AE1-B36 and P6TMU-TMUH-17). Correspondence: Dr. Shi-Chuan Chang, Chest Department, Taipei Veterans General Hospital, #201, Section 2, Shih-Pai Road, Shih-Pai, Taipei, Taiwan, 112 (E-mail: [email protected]).
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necrosis factor (TNF)-␣ and/or interleukin (IL)-1 in pleural fluid.7,8 These cytokines may reduce fibrinolytic activity by stimulating the release of PAI-1 and result in an imbalance between PAI-1 and tissue type plasminogen activator (tPA) in the pleural cavity. This imbalance leads to fibrin formation and deposition7–10 and subsequent loculation of the pleural effusion.11 These findings suggest a strong relationship between the fibrinolytic activity and proinflammatory cytokines in the pleural cavity. Clinically, patients with pleural transudates present with nonfibrinous, free-flowing effusions on chest ultrasonography.12 The levels of proinflammatory cytokines were significantly lower and fibrinolytic activity was significantly higher in pleural transudates than in exudates.13,14 Vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, increases vascular permeability and plays an important role in pleural fluid formation.15 In humans, pleural fluid VEGF levels are significantly higher in exudates than in transudates.16 Moreover, VEGF has been reported to regulate PA and PAI-1 activity in angiogenesis,17 and recently thought to play a pivotal role in the production of pleural fibrosis induced by transforming growth factor-2.18 However, its role in fibrinogenesis of the pleural space remains unclear. We hypothesize that repeated thoracenteses may cause pleural inflammation, induce local release of cytokines, and in turn enhance the release of PAI-1 leading to fibrin formation and deposition in transudative pleural effusions. In this study, we prospectively evaluated the effect of repeated thoracenteses on pleural fluid characteristics, the levels of inflammatory cytokines, and fibrinolytic activity in pleural transudates caused by heart failure and its clinical significance. Methods Patient Population The Institutional Review Board of Taipei Medical University Hospital approved this 2-year prospective study and informed consent was obtained from all patients participating in this study. The patients with symptomatic, large amount (greater than half of one hemithorax) free-flowing pleural effusion admitted for diagnostic evaluation between January 2004 and December 2005 were eligible for this study. All patients were examined within 24 hours of admission. Therapeutic thoracentesis with drainage of 500 mL of pleural fluid was performed under the guidance of chest ultrasonography. In the cases of bilateral pleural effusions, all thoracenteses were performed on the side with greater amount of fluid on presentation. The processing and analyses of pleural fluid were described previously.3 The patients were included if pleural transudate was established by the criteria of Light et al19 and was caused by congestive heart failure. A diagnosis of congestive heart failure was accepted with an appropriate clinical setting, radiographic features, depressed left ventricular function, response to therapy, and consistent course. The patients were excluded from this study if they had any of the following (1) invasive procedures directed into the pleural cavity or chest trauma within 3 months before hospitalization; (2) loculated pleural effusion or fibrin strands detected in pleural effusion by chest ultrasonography before the first thora-
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centesis; (3) use of diuretics before and during repeated thoracenteses; (4) diagnosis of renal failure, liver cirrhosis, collagen vascular diseases, or hematological disorders; (5) pleural effusion of undiagnosed cause. Repeated Thoracenteses and Detection of Fibrin Strands in Pleural Effusions The patients who met the inclusion criteria were subjected to repeated thoracenteses with drainage of 500 mL of pleural fluid at 24 hours (day 2) and 48 hours (day 3) after the first thoracentesis.3 During each thoracentesis, pleural fluid samples were collected and processed as the first thoracentesis. Chest ultrasonography was performed on the day 6 to observe the presence of fibrin strands in pleural effusion.3,4,12 Based on the presence or absence of fibrin strands in pleural effusions shown on chest ultrasonography on the day 6, the patients were classified into the fibrinous and nonfibrinous groups.3,4,12 Outcome of Pleural Effusion After the third thoracentesis, all patients were treated with diuretic therapy, including 40 to 80 mg/d of oral or intravenous furosemide. The clinical outcome of pleural effusion was evaluated 7 days after the third thoracentesis. Resolution was defined as no or minimal pleural effusion on chest radiography. Stationary indicated residual pleural effusion without causing clinical symptoms. Recurrence was defined as recurrence of symptomatic effusion requiring thoracentesis during the whole follow-up period. Measurement of Cytokines, tPA, and PAI-1 in Pleural Fluid The levels of cytokines, tPA, and PAI-1 in pleural fluid were measured using the following commercially available enzymelinked immunosorbent assay kits: tPA and PAI-1 (American Diagnostica, Greenwich, CT), TNF-␣, IL-1, IL-8, and VEGF (R&D Systems, Minneapolis, MN). Statistical Analysis Nonparametric tests were used to analyze pleural fluid variables since these variables were not normally distributed. Accordingly, the effect of repeated thoracenteses on the pleural fluid variables was analyzed using the Friedman test. Paired data comparisons were performed using a Wilcoxon signed-rank test. The correlations between variables were determined by Spearman rank correlation coefficients. Comparisons of continuous data between 2 groups were made using Student’s t test or Mann-Whitney U test when appropriate. Comparisons of categorical data between 2 groups were performed using 2 test and/or Fisher exact test. Significance was defined as P ⬍ 0.05 (twotailed). Statistical analysis was performed using a statistical software package (Statistica for Window, version 5.5, StatSoft Inc, Tulsa, OK).
Results Patient Characteristics A total of 21 patients with congestive heart failure were included in this study, including 14 men and 7 women with an age range from 65 to 90 years (mean, 82 years). All had shortness of breath, peripheral edema, and cardiomegaly (cardiothoracic index ⬎0.5). The mean ejection fraction was 43.0 ⫾ 3.5%. The causes of heart failure were hypertension (12 patients), coronary heart disease (8 patients), and valvular heart disease (1 patient). The pleural effusion was bilateral in 15 patients and unilateral in 6 patients. In all patients, the clinical symptoms improved after repeated thoracenteses. There was no evidence that traumatic thoracentesis occurred. 453
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Table 1. Changes of Pleural Fluid Characteristics During Repeated Thoracenteses in 21 Patients Effusion Variables
Day 1
Day 2
Day 3
Friedman Test P Value
pH Glucose, mg/dL Protein, g/dL LDH, IU/dL Erythrocytes, cells/L Total leukocytes, cells/L Neutrophils, cells/L Lymphocytes, cells/L Macrophages, cells/L Eosinophils, cells/L
7.53 (7.30–7.80) 153 (96–364) 2.1 (0.8–3.8) 73 (18–150) 360 (9–42000) 108 (0–1008) 47 (4–248) 39 (0–867) 19 (2–189) 2 (0–20)
7.49 (7.39–7.69) 154 (94–278) 2.2 (1.1–4.2) 139 (33–232)† 1512 (0–40000)* 162 (0–8000)† 91 (7–3680)† 75 (3–1248) 24 (5–130) 6 (0–113)
7.54 (7.30–7.80) 143 (90–308) 1.8 (1.0–4.1) 182 (62–556)†‡ 1458 (9–18000)* 180 (0–11520)† 59 (6–3802)† 57 (3–1912) 20 (5–419) 4 (0–106)
0.09 0.15 0.36 ⬍0.0001 0.039 0.002 0.002 0.053 0.65 0.32
* Values are presented as median (range). Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; LDH, lactate dehydrogenase. * P ⬍ 0.05 compared with day 1 value; † P ⬍ 0.01 compared with day 1 value; ‡ P ⬍ 0.01 compared with day 2 value.
There were no complications such as hemothorax, pneumothorax, reexpansion pulmonary edema, or sequential pleural infection. Pleural Fluid Characteristics Pleural fluid characteristics including erythrocytes, total leukocytes with differential, pH, lactate dehydrogenase (LDH), glucose, and protein obtained from day 1 to day 3 are shown in Table 1. Repeated thoracenteses had no effect on effusion pH, protein or glucose. In contrast, compared with the data from the first thoracentesis (day 1), significant increase in effusion LDH, erythrocytes, total leukocytes and neutrophils was found on day 2 and day 3. Furthermore, the levels of LDH on day 3 were significantly higher than those on day 2. Repeated thoracenteses did not affect other subpopulations of leukocytes (Table 1). Cytokines, tPA, and PAI-1 in Pleural Fluid The changes in effusion levels of cytokines, tPA, and PAI-1 from day 1 to day 3 are summarized in Table 2. The effusion levels of TNF-␣, IL-1, IL-8, VEGF, and PAI-1 increased significantly during repeated thoracenteses. The values of effusion TNF-␣, IL-1, IL-8, VEGF, and PAI-1 on day 2 and day 3 were significantly higher than those on day 1. More-
over, effusion levels of IL-1, VEGF, and PAI-1 on day 3 were significantly higher than those on day 2. In contrast, repeated thoracenteses had no effect on effusion tPA (Table 2). Correlation Between Effusion PAI-1, Ratio of PAI-1 to tPA, and Cytokines During repeated thoracenteses, effusion values of PAI-1 on day 2 and day 3 were highly correlated with those of TNF-␣, IL-1, IL-8, and VEGF (Table 3). Furthermore, the data of effusion PAI-1/tPA ratio measured on day 2 and day 3 were correlated positively with those of TNF-␣, IL-1, IL-8, and VEGF (Table 3). Fibrinous and Nonfibrinous Groups Chest ultrasonography was performed to observe the presence of fibrin strands in pleural effusion before each tap and on day 6 in all patients. Before thoracentesis, the chest ultrasonography showed large to massive amounts of free-flowing effusion in all patients (Figure 1A). On day 6, 6 of 21 patients (29%) showed fibrin strands in pleural effusions and were classified as the fibrinous group (Figure 1B). The remaining 15 patients who had no fibrin strands were classified as the nonfibrinous group.
Table 2. Changes of Effusion Cytokines, tPA, and PAI-1 During Repeated Thoracenteses in 21 Patients Variables
Day 1
Day 2
Day 3
Friedman Test P Value
TNF-␣, pg/mL IL-1, pg/mL IL-8, pg/mL VEGF, pg/mL PAI-1, ng/mL tPA, ng/mL
11.8 (1.5–27.4) 1.7 (1.3–3.4) 93.7 (50.0–678.2) 80.7 (39.7–1885.6) 38.3 (3.1–170.3) 12.5 (7.2–35.0)
14.4 (4.5–28.7)* 2.5 (1.3–5.3)† 163.5 (42.1–1264.3)† 147.9 (35.6–3945.1)* 65.7 (18.7–172.5)* 14.9 (8.2–35.2)
15.7 (4.5–56.7)* 2.7 (1.6–6.6)†‡ 190.6 (55.8–1208.1)† 257.9 (73.3–3168.5)†‡ 121.3 (18.7–180.9)†‡ 13.9 (8.3–35.1)
0.006 ⬍0.0001 0.003 0.0002 0.0001 0.26
Values are presented as median (range). Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin-1; IL-8, interleukin-8; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor type 1; tPA, tissue-type plasminogen activator. * P ⬍ 0.05 compared with day 1 value; † P ⬍ 0.01 compared with day 1 value; ‡ P ⬍ 0.01 compared with day 2 value.
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Table 3. Correlation Between Effusion PAI-1, Ratio of PAI-1 to tPA, and Cytokines During Repeated Thoracenteses in 21 Patients PAI-1
PAI-1/tPA
Variables
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
TNF-␣ IL-1 IL-8 VEGF
0.30 ⫺0.04 0.27 0.33
0.57† 0.45* 0.59† 0.72‡
0.48* 0.66† 0.46* 0.75‡
0.37 ⫺0.02 0.27 0.35
0.46* 0.49* 0.45* 0.56†
0.54* 0.55† 0.47* 0.50*
Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin-1; IL-8, interleukin-8; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor type 1; tPA, tissue type plasminogen activator. * Correlation is statistically significant at the level of 0.05. † Correlation is statistically significant at the level of 0.01. ‡ Correlation is statistically significant at the level of 0.001.
Comparisons Between Fibrinous and Nonfibrinous Groups All the fluid characteristics obtained during repeated thoracenteses were comparable between the fibrinous and nonfibrinous groups (data not shown). Compared with the nonfibrinous group, effusion levels of VEGF on day 1 were significantly higher in the fibrinous group. Furthermore, the effusion levels of TNF-␣, IL-1, VEGF, and PAI-1 on day 2 and day 3 were also significantly higher in the fibrinous group when compared with the nonfibrinous group (Figure 2, A through D). However, there was no significant difference in the pleural fluid levels of IL-8 and tPA between the 2 groups during repeated thoracenteses (data not shown). The clinical features and outcome of pleural effusions in fibrinous and nonfibrinous groups are summarized in Table 4. The left ventricular function and dose of diuretic given after the 3rd thoracentesis were comparable between the 2 groups. However, during the following 7 days after repeated thoracen-
Figure 1. An 80-year-old woman had right-sided massive pleural effusion. A, Before thoracentesis, chest ultrasonography performed on day 1 showed massive nonfibrinous effusion and collapsed lung (arrow). B, After repeated thoracenteses, chest ultrasonography performed on day 6 revealed residual pleural effusion, collapsed lung (arrow), and newly formed fibrin strands (arrowheads). E ⫽ effusion. L ⫽ liver. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
teses, the effusion resolution rate was significantly lower (0 of 6 vs 12 of 15, respectively, P ⫽ 0.002) and the effusion recurrence rate was significantly higher (3 of 6 vs 0 of 15, respectively, P ⫽ 0.015) in fibrinous than in nonfibrinous groups (Table 4). Discussion This is the first study to demonstrate a significant increase in the amounts of LDH, erythrocytes, total leukocytes, neutrophils, TNF-␣, IL-1, IL-8, VEGF, and PAI-1 in the pleural fluid after repeated thoracenteses in patients with transudative pleural effusions caused by congestive heart failure. The amounts of PAI-1 and PAI-1/tPA ratio on day 2 and day 3 in the pleural fluid were highly correlated with those of TNF-␣, IL1-, IL-8, and VEGF. On day 6, fibrin strands developed in pleural effusions shown on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of VEGF on day 1 and TNF-␣, IL-1, VEGF and PAI-1 on day 2 and day 3 were significantly higher in the fibrinous group. The effusion resolution rate was significantly lower and effusion recurrence rate was significantly higher in the fibrinous group than in the nonfibrinous group. In this study, we explored the effect of repeated thoracenteses on pleural fluid characteristics, cytokines and fibrinolytic activity in patients with transudative effusions caused by heart failure. Diuretics were withheld during the period of repeated thoracenteses to avoid any influence on the total protein level in transudative pleural fluid,20 which may confound the effect of repeated thoracenteses on effusion variables evaluated. Eventually, clinical symptoms and conditions improved in all patients after repeated thoracenteses. In line with our previous study on malignant pleural effusions,3 the current study revealed that significantly decreased fibrinolytic activity in pleural transudates during repeated thoracenteses might be regulated by the proinflammatory cytokines. Compared with the data on day 1, the effusion levels of TNF-␣ and IL-1 on day 2 and day 3 increased markedly as did PAI-1 and PAI-1/tPA ratio (Tables 2 and 3). Additionally, erythrocytes, total leukocytes, neutrophils, LDH, IL-8, and VEGF in pleural fluid were also significantly increased (Tables 1 and 2). Furthermore, PAI-1 and PAI-1/tPA ratio in the pleural fluid on day 2 and day 3 were highly correlated with those of IL-8 and VEGF (Table 3). It is well known that recruitment of circulating leukocytes to sites of inflammation is associated with angiogenesis. There is considerable evidence to suggest that angiogenesis and inflammation may occur in an interactive and overlapping manner. The 455
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Figure 2. Comparisons of effusion TNF-␣ (A), IL-1 (B), VEGF (C), and PAI-1 (D) during repeated thoracenteses between the fibrinous group (solid bars, n ⫽ 6) and the nonfibrinous group (open bars, n ⫽ 15). Box-and-whisker plots are based on the median, quartiles, and extreme values. Box represents the interquartile range that contains 50% of values. Whiskers are lines that extend from the box to the highest and lowest values.
increased neutrophils and total leukocytes in pleural fluid during repeated thoracenteses may be explained in part by an increase in IL-8, a chemokine for neutrophil recruitment into the pleural space.21 The increase of IL-8 in the pleural fluid during repeated thoracenteses may be involved in fibrinogenesis since IL-8 was reported to be responsible for activation of PAI-1 and blockage of fibrinolytic activity,22 and may also function as an angiogenic factor.23 VEGF is a potent inducer of vascular perTable 4. Comparison of Clinical Features and Outcome of Effusion Between Fibrinous and Nonfibrinous Groups Characteristics Mean Age, yr Gender, M/F, cases (%) Ejection fraction, % Furosemide dose after 3rd tap, mg/d Outcome of effusion† Resolution, No. (%) Stationary, No. (%) Recurrence, No. (%)
Fibrinous Group n ⫽ 6
Nonfibrinous Group n ⫽ 15
P Value
86 ⫾ 4 3/3 (50/50) 42 ⫾ 3 60 ⫾ 18
80 ⫾ 12 11/4 (73/27) 43 ⫾ 4 52 ⫾ 10
0.21 0.35 0.62 0.20
0 (0) 3 (50) 3 (50)
12 (75) 3 (25) 0 (0)
0.002 0.29 0.015
* Values are presented as mean ⫾ SD unless specified. † Values are presented as cumulative cases within 7 days after the third thoracentesis.
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meability and plays an important role in the formation of pleural fluid.15,24 –26 In addition, VEGF enhances permeability in the mesothelial monolayer.27 Furthermore, VEGF may indirectly stimulate recruitment of inflammatory cells,28 and regulate the production of PA and PAI-1 implicated in matrix degradation in angiogenesis.17 These findings suggest that VEGF may be involved in pleural inflammation and fibrinogenesis. After repeated thoracenteses, fibrin strands developed in the pleural fluid in 6 patients (fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). The presence of fibrin strands and significantly higher levels of TNF-␣, IL-1, VEGF and PAI-1 on day 2 and day 3, when compared with the nonfibrinous group, indicating that inflammation was enhanced and fibrinolytic activity was depressed during repeated thoracenteses in the pleural cavity in the fibrinous group (Figure 2, A through D). These findings further support that TNF-␣, IL-1, and VEGF may play a pivotal role in the regulation of fibrinolytic activity in transudative effusion during repeated thoracenteses. Taken together, the present study strongly suggests that repeated thoracenteses may cause pleural inflammation and induce local release of inflammatory cytokines and chemokines with subsequent recruitDecember 2007 Volume 334 Number 6
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ment of leukocytes, which in turn enhance local release of PAI-1 and lead to fibrin formation and deposition in the pleural cavity. With diuretic therapy, most transudative pleural effusions due to congestive heart failure resolved within 7 days.20 Accordingly, we evaluated the clinical outcome of transudative pleural effusion within 7 days after 3 consecutive thoracenteses. The current study revealed that the pleural effusion resolved successfully in only 12 of 21 patients. No recurrence of pleural effusion was found in the nonfibrinous group. In contrast, the pleural effusion failed to resolve in the fibrinous group, and 3 of the 6 patients had reaccumulation of pleural fluid (Table 4). These findings may be explained in part by the progressive increase of VEGF in the pleural fluid in sequential thoracenteses (Table 2) and significantly higher levels in the fibrinous group compared with the nonfibrinous group (Figure 2C). The elevated levels of VEGF may lead to an increase in the pleural vascular permeability and contribute to pleural fluid formation15,24 –26 and may thereby impede pleural fluid reabsorption. However, the number of subjects in this study was limited and further studies with larger sample size are needed to verify the role of repeated thoracenteses in the treatment of large to massive pleural transudates. In conclusion, the present study demonstrated that repeated thoracenteses might cause pleural inflammation and induce local release of proinflammatory cytokines, chemokines and VEGF, which might subsequently enhance the release of PAI-1 and lead to fibrin deposition in transudative effusions. These may impair the resolution of pleural effusions. References 1. Light RW. Transudative pleural effusions. In: Light RW, editor. Pleural Diseases. 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2001; p. 96–107. 2. Marel M, Zru ˚ stova´ M, Sˇtasny B, et al. The incidence of pleural effusion in a well-defined region. Chest 1993;104: 1486–9. 3. Chung CL, Chen YC, Chang SC. Effect of repeated thoracenteses on fluid characteristics, cytokines, and fibrinolytic activity in malignant pleural effusion. Chest 2003;123:1185–95. 4. McLoud TC, Flower CDR. Imaging the pleura: sonography, CT, and MR imaging. AJR Am J Roentgenol 1991;156:1145–53. 5. Bithell TC. Blood coagulation. In: Lee GR, Bithell TC, Foerster J, et al, editors. Wintrobe’s Clinical Hematology, 9th ed. Philadelphia: Lea & Febiger; 1993; p. 566–615. 6. Idell S, Zwieb C, Kumar A, et al. Pathways of fibrin turnover of human pleural mesothelial cells in vitro. Am J Respir Cell Mol Biol 1992;7:414–26. 7. Agrenius V, Chmielewska J, Widstro¨m O, et al. Pleural fibrinolytic activity is decreased in inflammation as demonstrated in quinacrine pleurodesis treatment of malignant pleural effusion. Am Rev Respir Dis 1989;140:1381–5. 8. Hua CC, Chang LC, Chen YC, et al. Proinflammatory cytokines and fibrinolytic enzymes in tuberculous and malignant pleural effusions. Chest 1999;116:1292–6.
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9. Whawell SA, Thompson JN. Cytokine-induced release of plasminogen activator inhibitor-1 by human mesothelial cells. Eur J Surg 1995;161:315–7. 10. Philip-Joe¨t F, Alessi MC, Philip-Joe¨t C, et al. Fibrinolytic and inflammatory processes in pleural effusions. Eur Respir J 1995;8:1352–6. 11. Chung CL, Chen CH, Sheu JR, et al. Proinflammatory cytokines, transforming growth factor-1, and fibrinolytic enzymes in loculated and free-flowing pleural exudates. Chest 2005;128:690–7. 12. Yang PC, Luh KT, Chang DB, et al. Value of sonography in determining the nature of pleural effusion: analysis of 320 cases. AJR Am J Roentgenol 1992;159:29–33. 13. Xirouchaki N, Tzanakis N, Bouros D, et al. Diagnostic value of interleukin-1␣, interleukin-6 and tumor necrosis factor in pleural effusions. Chest 2002;121:815–20. 14. Idell S, Girard W, Koenig KB, et al. Abnormality of pathways of fibrin turnover in the human pleural space. Am Rev Respir Dis 1991;144:187–94. 15. Grove CS, Lee YCG. Vascular endothelial growth factor: the key mediator in pleural effusion formation. Curr Opin Pulm Med 2002;8:294–301. 16. Cheng DS, Rodriguez RM, Perkett EA, et al. Vascular endothelial growth factor in pleural fluid. Chest 1999;116:760–5. 17. Pepper MS, Ferrara N, Orci L, et al. Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. Biochem Biophys Res Commun 1991;181:902–6. 18. Guo YB, Kalomenidis I, Hawthorne M, et al. Pleurodesis is inhibited by anti-vascular endothelial growth factor antibody. Chest 2005;128:1790–7. 19. Light RW, MacGregor MI, Luchsinger PC, et al. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77:507–13. 20. Romero-Candeira S, Ferna´ndez C, Martı´n C, et al. Influence of diuretics on the concentration of proteins and other components of pleural transudates in patients with heart failure. Am J Med 2001;110:681–6. 21. Broaddus VC, Hebert CA, Vitangcol RV, et al. Interleukin-8 is a major neutrophil chemotactic factor in pleural liquid of patients with empyema. Am Rev Respir Dis 1992;146:825–30. 22. Biemond BJ, Levi M, Ten Cate H, et al. Plasminogen activator and plasminogen activator inhibitor I release during experimental endotoxemia in chimpanzees: effect of intervention in cytokine and coagulation cascades. Clin Sci 1995;88:587–94. 23. Albini A, Tosetti F, Benelli R, et al. Tumor inflammatory angiogenesis and its chemoprevention. Cancer Res 2005;23: 10637–41. 24. Thickett DR, Armstrong L, Millar AB. Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions. Thorax 1999;54:707–10. 25. Yeo KT, Wang HH, Nagy JA, et al. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res 1993; 53:2912–8. 26. Lee YCG, Melderneker D, Thompson PJ, et al. Transforming growth factor  induces vascular endothelial growth factor elaboration from pleural mesothelial cells in vivo and in vitro. Am J Respir Crit Care Med 2002;165:88–94. 27. Mohammed KA, Nasreen N, Hardwick J, et al. Bacterial induction of pleural mesothelial monolayer barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 2001;281:L119–25. 28. Lee TH, Avraham H, Lee SH, et al. Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells. J Biol Chem 2002;277:10445–51.
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ABSTRACT: Background: Repeated thoracenteses is indicated in patients with refractory, symptomatic transudative effusions. However, their effect on cytokines and fibrinolytic activity in pleural transudates remains unclear. Methods: Twenty-one patients with symptomatic, large amount of free-flowing transudative effusions caused by heart failure were studied. Thoracentesis with drainage of 500 mL of pleural fluid per day was done for 3 consecutive days (days 1 to 3). Pleural fluid characteristics, tumor necrosis factor (TNF)-␣, interleukin (IL)1, IL-8, vascular endothelial growth factor (VEGF), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured during each tap. Chest ultrasonography was done on day 6 to detect the fibrin strands in pleural effusion and the outcome of effusion was evaluated within 7 days after repeated thoracenteses. Results: Effusion levels of lactate dehydrogenase, neutrophils, TNF-␣, IL-1, IL-8,
VEGF, and PAI-1 increased significantly during repeated thoracenteses. Furthermore, the values of PAI-1 and PAI-1/tPA obtained on days 2 and 3 were highly correlated with those of TNF-␣, IL-1, IL-8, and VEGF. On day 6, pleural fibrins were observed on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of TNF-␣, IL-1, VEGF, and PAI-1 on day 2 and day 3, and recurrence of symptomatic effusion after repeated thoracenteses were significantly higher in fibrinous group. Conclusions: Repeated thoracenteses may induce local release of proinflammatory cytokines, VEGF and PAI-1, which may result in fibrin deposition and impair resolution of pleural transudates. KEY INDEXING TERMS: Fibrinogenesis; Pleural effusion; Proinflammatory cytokine; Thoracentesis; Transudates. [Am J Med Sci 2007;334(6):452–457.]
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transudative effusion, which in general shows poor response to medical treatment.1 Occasionally, transudative pleural effusions may reaccumulate rapidly in patients with refractory heart failure and require repeated thoracenteses.1 Fibrinogenesis, defined as fibrin formation in the pleural cavity, induced by repeated thoracenteses, is not uncommon in pleural exudates as detected by chest ultrasonography.3,4 However, to the best of our knowledge, it remains unknown whether repeated thoracenteses can induce fibrinogenesis in transudative effusions, in which the pleura is considered to be intact and noninflammatory. Fibrin turnover in the pleural cavity is greatly affected by the activity of fibrinolysis. The formation of key enzyme in fibrinolysis, plasmin, is based on the equilibrium between plasminogen activators (PAs) and plasminogen activator inhibitors (PAIs).5 Fibrin formation and deposition are the hallmarks of pleural inflammation,6 which may enhance the release of proinflammatory cytokines such as tumor
ransudative pleural effusion occurs when hydrostatic and oncotic pressures across the pleural membrane are altered.1 Congestive heart failure is the most common cause of transudative pleural effusions, which may aggravate the dyspnea in these patients.2 Therapeutic thoracentesis may be indicated, particularly in those with a large amount of
From the Department of Chest Medicine, Taipei Medical University Hospital (CLC, CYY); Graduate Institute of Clinical Medicine (CLC); Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University (JRS); Chest Department, Taipei Veterans General Hospital (YCC, SCC); and Institute of Emergency and Critical Care Medicine (SCC), School of Medicine, National Yang-Ming University, Taipei, Taiwan. Submitted March 15, 2007; accepted in revised form May 22, 2007. This work was supported by grants from the Taipei Medical University (TMU92-AE1-B36 and P6TMU-TMUH-17). Correspondence: Dr. Shi-Chuan Chang, Chest Department, Taipei Veterans General Hospital, #201, Section 2, Shih-Pai Road, Shih-Pai, Taipei, Taiwan, 112 (E-mail: [email protected]).
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necrosis factor (TNF)-␣ and/or interleukin (IL)-1 in pleural fluid.7,8 These cytokines may reduce fibrinolytic activity by stimulating the release of PAI-1 and result in an imbalance between PAI-1 and tissue type plasminogen activator (tPA) in the pleural cavity. This imbalance leads to fibrin formation and deposition7–10 and subsequent loculation of the pleural effusion.11 These findings suggest a strong relationship between the fibrinolytic activity and proinflammatory cytokines in the pleural cavity. Clinically, patients with pleural transudates present with nonfibrinous, free-flowing effusions on chest ultrasonography.12 The levels of proinflammatory cytokines were significantly lower and fibrinolytic activity was significantly higher in pleural transudates than in exudates.13,14 Vascular endothelial growth factor (VEGF), a potent angiogenic cytokine, increases vascular permeability and plays an important role in pleural fluid formation.15 In humans, pleural fluid VEGF levels are significantly higher in exudates than in transudates.16 Moreover, VEGF has been reported to regulate PA and PAI-1 activity in angiogenesis,17 and recently thought to play a pivotal role in the production of pleural fibrosis induced by transforming growth factor-2.18 However, its role in fibrinogenesis of the pleural space remains unclear. We hypothesize that repeated thoracenteses may cause pleural inflammation, induce local release of cytokines, and in turn enhance the release of PAI-1 leading to fibrin formation and deposition in transudative pleural effusions. In this study, we prospectively evaluated the effect of repeated thoracenteses on pleural fluid characteristics, the levels of inflammatory cytokines, and fibrinolytic activity in pleural transudates caused by heart failure and its clinical significance. Methods Patient Population The Institutional Review Board of Taipei Medical University Hospital approved this 2-year prospective study and informed consent was obtained from all patients participating in this study. The patients with symptomatic, large amount (greater than half of one hemithorax) free-flowing pleural effusion admitted for diagnostic evaluation between January 2004 and December 2005 were eligible for this study. All patients were examined within 24 hours of admission. Therapeutic thoracentesis with drainage of 500 mL of pleural fluid was performed under the guidance of chest ultrasonography. In the cases of bilateral pleural effusions, all thoracenteses were performed on the side with greater amount of fluid on presentation. The processing and analyses of pleural fluid were described previously.3 The patients were included if pleural transudate was established by the criteria of Light et al19 and was caused by congestive heart failure. A diagnosis of congestive heart failure was accepted with an appropriate clinical setting, radiographic features, depressed left ventricular function, response to therapy, and consistent course. The patients were excluded from this study if they had any of the following (1) invasive procedures directed into the pleural cavity or chest trauma within 3 months before hospitalization; (2) loculated pleural effusion or fibrin strands detected in pleural effusion by chest ultrasonography before the first thora-
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centesis; (3) use of diuretics before and during repeated thoracenteses; (4) diagnosis of renal failure, liver cirrhosis, collagen vascular diseases, or hematological disorders; (5) pleural effusion of undiagnosed cause. Repeated Thoracenteses and Detection of Fibrin Strands in Pleural Effusions The patients who met the inclusion criteria were subjected to repeated thoracenteses with drainage of 500 mL of pleural fluid at 24 hours (day 2) and 48 hours (day 3) after the first thoracentesis.3 During each thoracentesis, pleural fluid samples were collected and processed as the first thoracentesis. Chest ultrasonography was performed on the day 6 to observe the presence of fibrin strands in pleural effusion.3,4,12 Based on the presence or absence of fibrin strands in pleural effusions shown on chest ultrasonography on the day 6, the patients were classified into the fibrinous and nonfibrinous groups.3,4,12 Outcome of Pleural Effusion After the third thoracentesis, all patients were treated with diuretic therapy, including 40 to 80 mg/d of oral or intravenous furosemide. The clinical outcome of pleural effusion was evaluated 7 days after the third thoracentesis. Resolution was defined as no or minimal pleural effusion on chest radiography. Stationary indicated residual pleural effusion without causing clinical symptoms. Recurrence was defined as recurrence of symptomatic effusion requiring thoracentesis during the whole follow-up period. Measurement of Cytokines, tPA, and PAI-1 in Pleural Fluid The levels of cytokines, tPA, and PAI-1 in pleural fluid were measured using the following commercially available enzymelinked immunosorbent assay kits: tPA and PAI-1 (American Diagnostica, Greenwich, CT), TNF-␣, IL-1, IL-8, and VEGF (R&D Systems, Minneapolis, MN). Statistical Analysis Nonparametric tests were used to analyze pleural fluid variables since these variables were not normally distributed. Accordingly, the effect of repeated thoracenteses on the pleural fluid variables was analyzed using the Friedman test. Paired data comparisons were performed using a Wilcoxon signed-rank test. The correlations between variables were determined by Spearman rank correlation coefficients. Comparisons of continuous data between 2 groups were made using Student’s t test or Mann-Whitney U test when appropriate. Comparisons of categorical data between 2 groups were performed using 2 test and/or Fisher exact test. Significance was defined as P ⬍ 0.05 (twotailed). Statistical analysis was performed using a statistical software package (Statistica for Window, version 5.5, StatSoft Inc, Tulsa, OK).
Results Patient Characteristics A total of 21 patients with congestive heart failure were included in this study, including 14 men and 7 women with an age range from 65 to 90 years (mean, 82 years). All had shortness of breath, peripheral edema, and cardiomegaly (cardiothoracic index ⬎0.5). The mean ejection fraction was 43.0 ⫾ 3.5%. The causes of heart failure were hypertension (12 patients), coronary heart disease (8 patients), and valvular heart disease (1 patient). The pleural effusion was bilateral in 15 patients and unilateral in 6 patients. In all patients, the clinical symptoms improved after repeated thoracenteses. There was no evidence that traumatic thoracentesis occurred. 453
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Table 1. Changes of Pleural Fluid Characteristics During Repeated Thoracenteses in 21 Patients Effusion Variables
Day 1
Day 2
Day 3
Friedman Test P Value
pH Glucose, mg/dL Protein, g/dL LDH, IU/dL Erythrocytes, cells/L Total leukocytes, cells/L Neutrophils, cells/L Lymphocytes, cells/L Macrophages, cells/L Eosinophils, cells/L
7.53 (7.30–7.80) 153 (96–364) 2.1 (0.8–3.8) 73 (18–150) 360 (9–42000) 108 (0–1008) 47 (4–248) 39 (0–867) 19 (2–189) 2 (0–20)
7.49 (7.39–7.69) 154 (94–278) 2.2 (1.1–4.2) 139 (33–232)† 1512 (0–40000)* 162 (0–8000)† 91 (7–3680)† 75 (3–1248) 24 (5–130) 6 (0–113)
7.54 (7.30–7.80) 143 (90–308) 1.8 (1.0–4.1) 182 (62–556)†‡ 1458 (9–18000)* 180 (0–11520)† 59 (6–3802)† 57 (3–1912) 20 (5–419) 4 (0–106)
0.09 0.15 0.36 ⬍0.0001 0.039 0.002 0.002 0.053 0.65 0.32
* Values are presented as median (range). Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; LDH, lactate dehydrogenase. * P ⬍ 0.05 compared with day 1 value; † P ⬍ 0.01 compared with day 1 value; ‡ P ⬍ 0.01 compared with day 2 value.
There were no complications such as hemothorax, pneumothorax, reexpansion pulmonary edema, or sequential pleural infection. Pleural Fluid Characteristics Pleural fluid characteristics including erythrocytes, total leukocytes with differential, pH, lactate dehydrogenase (LDH), glucose, and protein obtained from day 1 to day 3 are shown in Table 1. Repeated thoracenteses had no effect on effusion pH, protein or glucose. In contrast, compared with the data from the first thoracentesis (day 1), significant increase in effusion LDH, erythrocytes, total leukocytes and neutrophils was found on day 2 and day 3. Furthermore, the levels of LDH on day 3 were significantly higher than those on day 2. Repeated thoracenteses did not affect other subpopulations of leukocytes (Table 1). Cytokines, tPA, and PAI-1 in Pleural Fluid The changes in effusion levels of cytokines, tPA, and PAI-1 from day 1 to day 3 are summarized in Table 2. The effusion levels of TNF-␣, IL-1, IL-8, VEGF, and PAI-1 increased significantly during repeated thoracenteses. The values of effusion TNF-␣, IL-1, IL-8, VEGF, and PAI-1 on day 2 and day 3 were significantly higher than those on day 1. More-
over, effusion levels of IL-1, VEGF, and PAI-1 on day 3 were significantly higher than those on day 2. In contrast, repeated thoracenteses had no effect on effusion tPA (Table 2). Correlation Between Effusion PAI-1, Ratio of PAI-1 to tPA, and Cytokines During repeated thoracenteses, effusion values of PAI-1 on day 2 and day 3 were highly correlated with those of TNF-␣, IL-1, IL-8, and VEGF (Table 3). Furthermore, the data of effusion PAI-1/tPA ratio measured on day 2 and day 3 were correlated positively with those of TNF-␣, IL-1, IL-8, and VEGF (Table 3). Fibrinous and Nonfibrinous Groups Chest ultrasonography was performed to observe the presence of fibrin strands in pleural effusion before each tap and on day 6 in all patients. Before thoracentesis, the chest ultrasonography showed large to massive amounts of free-flowing effusion in all patients (Figure 1A). On day 6, 6 of 21 patients (29%) showed fibrin strands in pleural effusions and were classified as the fibrinous group (Figure 1B). The remaining 15 patients who had no fibrin strands were classified as the nonfibrinous group.
Table 2. Changes of Effusion Cytokines, tPA, and PAI-1 During Repeated Thoracenteses in 21 Patients Variables
Day 1
Day 2
Day 3
Friedman Test P Value
TNF-␣, pg/mL IL-1, pg/mL IL-8, pg/mL VEGF, pg/mL PAI-1, ng/mL tPA, ng/mL
11.8 (1.5–27.4) 1.7 (1.3–3.4) 93.7 (50.0–678.2) 80.7 (39.7–1885.6) 38.3 (3.1–170.3) 12.5 (7.2–35.0)
14.4 (4.5–28.7)* 2.5 (1.3–5.3)† 163.5 (42.1–1264.3)† 147.9 (35.6–3945.1)* 65.7 (18.7–172.5)* 14.9 (8.2–35.2)
15.7 (4.5–56.7)* 2.7 (1.6–6.6)†‡ 190.6 (55.8–1208.1)† 257.9 (73.3–3168.5)†‡ 121.3 (18.7–180.9)†‡ 13.9 (8.3–35.1)
0.006 ⬍0.0001 0.003 0.0002 0.0001 0.26
Values are presented as median (range). Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin-1; IL-8, interleukin-8; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor type 1; tPA, tissue-type plasminogen activator. * P ⬍ 0.05 compared with day 1 value; † P ⬍ 0.01 compared with day 1 value; ‡ P ⬍ 0.01 compared with day 2 value.
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Table 3. Correlation Between Effusion PAI-1, Ratio of PAI-1 to tPA, and Cytokines During Repeated Thoracenteses in 21 Patients PAI-1
PAI-1/tPA
Variables
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
TNF-␣ IL-1 IL-8 VEGF
0.30 ⫺0.04 0.27 0.33
0.57† 0.45* 0.59† 0.72‡
0.48* 0.66† 0.46* 0.75‡
0.37 ⫺0.02 0.27 0.35
0.46* 0.49* 0.45* 0.56†
0.54* 0.55† 0.47* 0.50*
Day 1, First thoracentesis; day 2, 24 hours after first thoracentesis; day 3, 48 hours after first thoracentesis; TNF-␣, tumor necrosis factor-␣; IL-1, interleukin-1; IL-8, interleukin-8; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor type 1; tPA, tissue type plasminogen activator. * Correlation is statistically significant at the level of 0.05. † Correlation is statistically significant at the level of 0.01. ‡ Correlation is statistically significant at the level of 0.001.
Comparisons Between Fibrinous and Nonfibrinous Groups All the fluid characteristics obtained during repeated thoracenteses were comparable between the fibrinous and nonfibrinous groups (data not shown). Compared with the nonfibrinous group, effusion levels of VEGF on day 1 were significantly higher in the fibrinous group. Furthermore, the effusion levels of TNF-␣, IL-1, VEGF, and PAI-1 on day 2 and day 3 were also significantly higher in the fibrinous group when compared with the nonfibrinous group (Figure 2, A through D). However, there was no significant difference in the pleural fluid levels of IL-8 and tPA between the 2 groups during repeated thoracenteses (data not shown). The clinical features and outcome of pleural effusions in fibrinous and nonfibrinous groups are summarized in Table 4. The left ventricular function and dose of diuretic given after the 3rd thoracentesis were comparable between the 2 groups. However, during the following 7 days after repeated thoracen-
Figure 1. An 80-year-old woman had right-sided massive pleural effusion. A, Before thoracentesis, chest ultrasonography performed on day 1 showed massive nonfibrinous effusion and collapsed lung (arrow). B, After repeated thoracenteses, chest ultrasonography performed on day 6 revealed residual pleural effusion, collapsed lung (arrow), and newly formed fibrin strands (arrowheads). E ⫽ effusion. L ⫽ liver. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
teses, the effusion resolution rate was significantly lower (0 of 6 vs 12 of 15, respectively, P ⫽ 0.002) and the effusion recurrence rate was significantly higher (3 of 6 vs 0 of 15, respectively, P ⫽ 0.015) in fibrinous than in nonfibrinous groups (Table 4). Discussion This is the first study to demonstrate a significant increase in the amounts of LDH, erythrocytes, total leukocytes, neutrophils, TNF-␣, IL-1, IL-8, VEGF, and PAI-1 in the pleural fluid after repeated thoracenteses in patients with transudative pleural effusions caused by congestive heart failure. The amounts of PAI-1 and PAI-1/tPA ratio on day 2 and day 3 in the pleural fluid were highly correlated with those of TNF-␣, IL1-, IL-8, and VEGF. On day 6, fibrin strands developed in pleural effusions shown on chest ultrasonography in 6 patients (29%, fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). Compared with the nonfibrinous group, the effusion levels of VEGF on day 1 and TNF-␣, IL-1, VEGF and PAI-1 on day 2 and day 3 were significantly higher in the fibrinous group. The effusion resolution rate was significantly lower and effusion recurrence rate was significantly higher in the fibrinous group than in the nonfibrinous group. In this study, we explored the effect of repeated thoracenteses on pleural fluid characteristics, cytokines and fibrinolytic activity in patients with transudative effusions caused by heart failure. Diuretics were withheld during the period of repeated thoracenteses to avoid any influence on the total protein level in transudative pleural fluid,20 which may confound the effect of repeated thoracenteses on effusion variables evaluated. Eventually, clinical symptoms and conditions improved in all patients after repeated thoracenteses. In line with our previous study on malignant pleural effusions,3 the current study revealed that significantly decreased fibrinolytic activity in pleural transudates during repeated thoracenteses might be regulated by the proinflammatory cytokines. Compared with the data on day 1, the effusion levels of TNF-␣ and IL-1 on day 2 and day 3 increased markedly as did PAI-1 and PAI-1/tPA ratio (Tables 2 and 3). Additionally, erythrocytes, total leukocytes, neutrophils, LDH, IL-8, and VEGF in pleural fluid were also significantly increased (Tables 1 and 2). Furthermore, PAI-1 and PAI-1/tPA ratio in the pleural fluid on day 2 and day 3 were highly correlated with those of IL-8 and VEGF (Table 3). It is well known that recruitment of circulating leukocytes to sites of inflammation is associated with angiogenesis. There is considerable evidence to suggest that angiogenesis and inflammation may occur in an interactive and overlapping manner. The 455
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Figure 2. Comparisons of effusion TNF-␣ (A), IL-1 (B), VEGF (C), and PAI-1 (D) during repeated thoracenteses between the fibrinous group (solid bars, n ⫽ 6) and the nonfibrinous group (open bars, n ⫽ 15). Box-and-whisker plots are based on the median, quartiles, and extreme values. Box represents the interquartile range that contains 50% of values. Whiskers are lines that extend from the box to the highest and lowest values.
increased neutrophils and total leukocytes in pleural fluid during repeated thoracenteses may be explained in part by an increase in IL-8, a chemokine for neutrophil recruitment into the pleural space.21 The increase of IL-8 in the pleural fluid during repeated thoracenteses may be involved in fibrinogenesis since IL-8 was reported to be responsible for activation of PAI-1 and blockage of fibrinolytic activity,22 and may also function as an angiogenic factor.23 VEGF is a potent inducer of vascular perTable 4. Comparison of Clinical Features and Outcome of Effusion Between Fibrinous and Nonfibrinous Groups Characteristics Mean Age, yr Gender, M/F, cases (%) Ejection fraction, % Furosemide dose after 3rd tap, mg/d Outcome of effusion† Resolution, No. (%) Stationary, No. (%) Recurrence, No. (%)
Fibrinous Group n ⫽ 6
Nonfibrinous Group n ⫽ 15
P Value
86 ⫾ 4 3/3 (50/50) 42 ⫾ 3 60 ⫾ 18
80 ⫾ 12 11/4 (73/27) 43 ⫾ 4 52 ⫾ 10
0.21 0.35 0.62 0.20
0 (0) 3 (50) 3 (50)
12 (75) 3 (25) 0 (0)
0.002 0.29 0.015
* Values are presented as mean ⫾ SD unless specified. † Values are presented as cumulative cases within 7 days after the third thoracentesis.
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meability and plays an important role in the formation of pleural fluid.15,24 –26 In addition, VEGF enhances permeability in the mesothelial monolayer.27 Furthermore, VEGF may indirectly stimulate recruitment of inflammatory cells,28 and regulate the production of PA and PAI-1 implicated in matrix degradation in angiogenesis.17 These findings suggest that VEGF may be involved in pleural inflammation and fibrinogenesis. After repeated thoracenteses, fibrin strands developed in the pleural fluid in 6 patients (fibrinous group) but were absent in the remaining 15 patients (nonfibrinous group). The presence of fibrin strands and significantly higher levels of TNF-␣, IL-1, VEGF and PAI-1 on day 2 and day 3, when compared with the nonfibrinous group, indicating that inflammation was enhanced and fibrinolytic activity was depressed during repeated thoracenteses in the pleural cavity in the fibrinous group (Figure 2, A through D). These findings further support that TNF-␣, IL-1, and VEGF may play a pivotal role in the regulation of fibrinolytic activity in transudative effusion during repeated thoracenteses. Taken together, the present study strongly suggests that repeated thoracenteses may cause pleural inflammation and induce local release of inflammatory cytokines and chemokines with subsequent recruitDecember 2007 Volume 334 Number 6
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ment of leukocytes, which in turn enhance local release of PAI-1 and lead to fibrin formation and deposition in the pleural cavity. With diuretic therapy, most transudative pleural effusions due to congestive heart failure resolved within 7 days.20 Accordingly, we evaluated the clinical outcome of transudative pleural effusion within 7 days after 3 consecutive thoracenteses. The current study revealed that the pleural effusion resolved successfully in only 12 of 21 patients. No recurrence of pleural effusion was found in the nonfibrinous group. In contrast, the pleural effusion failed to resolve in the fibrinous group, and 3 of the 6 patients had reaccumulation of pleural fluid (Table 4). These findings may be explained in part by the progressive increase of VEGF in the pleural fluid in sequential thoracenteses (Table 2) and significantly higher levels in the fibrinous group compared with the nonfibrinous group (Figure 2C). The elevated levels of VEGF may lead to an increase in the pleural vascular permeability and contribute to pleural fluid formation15,24 –26 and may thereby impede pleural fluid reabsorption. However, the number of subjects in this study was limited and further studies with larger sample size are needed to verify the role of repeated thoracenteses in the treatment of large to massive pleural transudates. In conclusion, the present study demonstrated that repeated thoracenteses might cause pleural inflammation and induce local release of proinflammatory cytokines, chemokines and VEGF, which might subsequently enhance the release of PAI-1 and lead to fibrin deposition in transudative effusions. These may impair the resolution of pleural effusions. References 1. Light RW. Transudative pleural effusions. In: Light RW, editor. Pleural Diseases. 4th ed. Philadelphia: Lippincott Williams & Wilkins; 2001; p. 96–107. 2. Marel M, Zru ˚ stova´ M, Sˇtasny B, et al. The incidence of pleural effusion in a well-defined region. Chest 1993;104: 1486–9. 3. Chung CL, Chen YC, Chang SC. Effect of repeated thoracenteses on fluid characteristics, cytokines, and fibrinolytic activity in malignant pleural effusion. Chest 2003;123:1185–95. 4. McLoud TC, Flower CDR. Imaging the pleura: sonography, CT, and MR imaging. AJR Am J Roentgenol 1991;156:1145–53. 5. Bithell TC. Blood coagulation. In: Lee GR, Bithell TC, Foerster J, et al, editors. Wintrobe’s Clinical Hematology, 9th ed. Philadelphia: Lea & Febiger; 1993; p. 566–615. 6. Idell S, Zwieb C, Kumar A, et al. Pathways of fibrin turnover of human pleural mesothelial cells in vitro. Am J Respir Cell Mol Biol 1992;7:414–26. 7. Agrenius V, Chmielewska J, Widstro¨m O, et al. Pleural fibrinolytic activity is decreased in inflammation as demonstrated in quinacrine pleurodesis treatment of malignant pleural effusion. Am Rev Respir Dis 1989;140:1381–5. 8. Hua CC, Chang LC, Chen YC, et al. Proinflammatory cytokines and fibrinolytic enzymes in tuberculous and malignant pleural effusions. Chest 1999;116:1292–6.
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9. Whawell SA, Thompson JN. Cytokine-induced release of plasminogen activator inhibitor-1 by human mesothelial cells. Eur J Surg 1995;161:315–7. 10. Philip-Joe¨t F, Alessi MC, Philip-Joe¨t C, et al. Fibrinolytic and inflammatory processes in pleural effusions. Eur Respir J 1995;8:1352–6. 11. Chung CL, Chen CH, Sheu JR, et al. Proinflammatory cytokines, transforming growth factor-1, and fibrinolytic enzymes in loculated and free-flowing pleural exudates. Chest 2005;128:690–7. 12. Yang PC, Luh KT, Chang DB, et al. Value of sonography in determining the nature of pleural effusion: analysis of 320 cases. AJR Am J Roentgenol 1992;159:29–33. 13. Xirouchaki N, Tzanakis N, Bouros D, et al. Diagnostic value of interleukin-1␣, interleukin-6 and tumor necrosis factor in pleural effusions. Chest 2002;121:815–20. 14. Idell S, Girard W, Koenig KB, et al. Abnormality of pathways of fibrin turnover in the human pleural space. Am Rev Respir Dis 1991;144:187–94. 15. Grove CS, Lee YCG. Vascular endothelial growth factor: the key mediator in pleural effusion formation. Curr Opin Pulm Med 2002;8:294–301. 16. Cheng DS, Rodriguez RM, Perkett EA, et al. Vascular endothelial growth factor in pleural fluid. Chest 1999;116:760–5. 17. Pepper MS, Ferrara N, Orci L, et al. Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells. Biochem Biophys Res Commun 1991;181:902–6. 18. Guo YB, Kalomenidis I, Hawthorne M, et al. Pleurodesis is inhibited by anti-vascular endothelial growth factor antibody. Chest 2005;128:1790–7. 19. Light RW, MacGregor MI, Luchsinger PC, et al. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77:507–13. 20. Romero-Candeira S, Ferna´ndez C, Martı´n C, et al. Influence of diuretics on the concentration of proteins and other components of pleural transudates in patients with heart failure. Am J Med 2001;110:681–6. 21. Broaddus VC, Hebert CA, Vitangcol RV, et al. Interleukin-8 is a major neutrophil chemotactic factor in pleural liquid of patients with empyema. Am Rev Respir Dis 1992;146:825–30. 22. Biemond BJ, Levi M, Ten Cate H, et al. Plasminogen activator and plasminogen activator inhibitor I release during experimental endotoxemia in chimpanzees: effect of intervention in cytokine and coagulation cascades. Clin Sci 1995;88:587–94. 23. Albini A, Tosetti F, Benelli R, et al. Tumor inflammatory angiogenesis and its chemoprevention. Cancer Res 2005;23: 10637–41. 24. Thickett DR, Armstrong L, Millar AB. Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions. Thorax 1999;54:707–10. 25. Yeo KT, Wang HH, Nagy JA, et al. Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions. Cancer Res 1993; 53:2912–8. 26. Lee YCG, Melderneker D, Thompson PJ, et al. Transforming growth factor  induces vascular endothelial growth factor elaboration from pleural mesothelial cells in vivo and in vitro. Am J Respir Crit Care Med 2002;165:88–94. 27. Mohammed KA, Nasreen N, Hardwick J, et al. Bacterial induction of pleural mesothelial monolayer barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 2001;281:L119–25. 28. Lee TH, Avraham H, Lee SH, et al. Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells. J Biol Chem 2002;277:10445–51.
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