ORIGINAL ARTICLE
Monoclonal anti-vascular endothelial growth factor antibody reduces fluid volume in an experimental model of inflammatory pleural effusion resp_1628
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SABRINA C.C. RIBEIRO, FRANCISCO S. VARGAS, LEILA ANTONANGELO, EVALDO MARCHI, EDUARDO H. GENOFRE, MILENA M.P. ACENCIO AND LISETE R. TEIXEIRA Laboratory of Pleura, Pulmonary Division, Heart Institute (InCor), University of Sao Paulo Medical School, Sao Paulo, Brazil
ABSTRACT
SUMMARY AT A GLANCE
Background and objective: Vascular endothelial growth factor (VEGF) is known to increase vascular permeability and promote angiogenesis. It is expressed in most types of pleural effusions. However, the exact role of VEGF in the development of pleural effusions has yet to be determined. The anti-VEGF mAb, bevacizumab, has been used in the treatment of cancer to reduce local angiogenesis and tumour progression. This study describes the acute effects of VEGF blockade on the expression of inflammatory cytokines and pleural fluid accumulation. Methods: One hundred and twelve New Zealand rabbits received intrapleural injections of either talc or silver nitrate. In each group, half the animals received an intravenous injection of bevacizumab, 30 min before the intrapleural agent was administered. Five animals from each subgroup were sacrificed 1, 2, 3, 4 or 7 days after the procedure. Twelve rabbits were used to evaluate vascular permeability using Evans’s blue dye. Pleural fluid volume and cytokines were quantified. Results: Animals pretreated with anti-VEGF antibody showed significant reductions in pleural fluid volumes after talc or silver nitrate injection. IL-8 levels, vascular permeability and macroscopic pleural adhesion scores were also reduced in the groups that received bevacizumab. Conclusions: This study showed that bevacizumab interferes in the acute phase of pleural inflammation induced by silver nitrate or talc, reinforcing the role of VEGF as a key mediator in the production of pleural effusions. The results also suggest that bevacizumab should probably be avoided in patients requiring pleurodesis.
The influence of vascular endothelial growth factor (VEGF) on pleural inflammation induced by sclerosants was evaluated. Anti-VEGF mAb reduced pleural fluid volume, IL-8 levels, vascular permeability and macroscopic adhesion scores. These data reinforce the importance of VEGF in production of pleural fluid and suggest that anti-VEGF agents should be avoided in patients requiring pleurodesis.
INTRODUCTION
Correspondence: Sabrina C.C. Ribeiro, Rua Arruda Alvim 107 ap 142 Pinheiros S. Paulo, 05410-020, Brazil. Email:
[email protected] Received 17 February 2009; invited to revise 26 March 2009, 7 June 2009; revised 1 June 2009, 11 June 2009; accepted 12 June 2009 (Associate Editor: Jose Porcel).
Intrapleural injection of sclerosing agents is commonly used to treat recurrent pleural effusions, especially those of malignant origin.1 Among the agents injected into the pleural space, talc and silver nitrate are widely used because of their ease of handling, low cost and high efficacy in both humans and animals.2–5 The injection of either silver nitrate or talc results in acute inflammation of the pleural cavity, producing a significant amount of pleural fluid, which is rich in proteins and LDH. Several cytokines are also produced in pleural fluid and serum, including IL-8, as well as growth factors such as vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-b). Although the pathophysiological mechanisms involved are not completely understood, it is believed that this inflammatory response precedes the events which will result in fibrosis and consequent pleural symphysis.6,7 Vascular endothelial growth factor is considered the main modulator of angiogenesis. It is much more potent than histamine in increasing vascular permeability, and has also been demonstrated to induce fenestrations in endothelial cells both in vivo and in vitro. It is believed to be one of the factors responsible for the formation of pleural effusions.8–12 In this context, the objective of the present study was to evaluate the influence of intravenous anti-VEGF mAb,
© 2009 The Authors Journal compilation © 2009 Asian Pacific Society of Respirology
Respirology (2009) 14, 1188–1193 doi: 10.1111/j.1440-1843.2009.01628.x
Key words: bevacizumab, cytokines, inflammation, pleural, effusion, silver, nitrate, talc.
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bevacizumab, in the acute phase of pleural inflammation induced by talc or silver nitrate.
METHODS Animals A total of 112 white New Zealand rabbits (2.0–2.5 kg) were used in the study, which was approved by the Ethics Committee of the Clinical Hospital, University of Sao Paulo Medical School. Using previously described methods,3,13–15 100 rabbits were divided into four subgroups of 25 animals each. After anaesthesia by intramuscular injection of 35 mg/kg of ketamine hydrochloride (Cristalia, Sao Paulo, Brazil) and 5 mg/kg of xylazine hydrochloride (Bayer, Sao Paulo, Brazil), trichotomy of the chest was performed and the skin was cleaned with 70% alcohol. Next, the animals were placed on a surgical table and a 21-G needle was inserted into the right pleural space for injection of 2 mL of 0.5% silver nitrate (Merck, Darmstadt, Germany) or 400 mg/kg of talc (USP Pharmacy, Sao Paulo, Brazil). Two of the four subgroups received an intravenous injection of 5 mg/kg of bevacizumab (Avastin, Roche, Basel, Switzerland) 30 min prior to the administration of the sclerosant. The animals that received bevacizumab were regarded as the treated group, while those that received only the intrapleural agents (silver nitrate or talc) were regarded as nontreated. The left pleural cavity was not manipulated and was used as a control for macroscopic and microscopic analyses.
Evaluation of pleural fluid and tissues Five animals from each subgroup were euthanized by intravenous injection of pentobarbital 1, 2, 3, 4 or 7 days after the procedure. A midline abdominal incision was made and a 19-G needle was introduced through the diaphragm in order to empty the pleural cavity and quantify the volume of pleural fluid. The chest was removed en bloc and the lungs expanded and fixed with 10% formalin. After a minimum period of 48 h, the pleural cavity was exposed for macroscopic examination. Adhesions were evaluated semi-quantitatively by two blinded examiners (L.T.R. and S.C.C.R.) using scores from 0 to 4 as follows: (0) normal pleural space; (1) one to three small adhesions; (2) more than three adhesions, with the lung being easily separated from the chest; (3) generalized adhesions, with areas of difficult separation between the lung and thoracic wall; and (4) complete obliteration of the pleural space. Samples of the visceral pleura were fixed and stained with haematoxylin-eosin and evaluated for the presence of inflammation and fibrosis. Microscopic analysis was performed by a pathologist (L.A.), who was blinded to the treatment. Inflammation and fibrosis were graded with a score from 0 to 4, according to the intensity and extent of pleural compromise as follows: (0) normal pleura or parenchyma; (1) © 2009 The Authors Journal compilation © 2009 Asian Pacific Society of Respirology
equivocal; (2) mild changes; (3) moderate changes; and (4) marked changes.14,15 Pleural fluid was collected in ethylenediamine tetra-acetic acid for cytological and cytokine analyses, and in untreated tubes for analysis of total protein and LDH. Pleural fluid was placed in a haemocytometer for quantification of total cells and onto slides for differential leucocyte counts (Leishman staining). Additional samples were centrifuged at 1000 rpm for 10 min at 4°C for protein and LDH analyses, and stored at -80°C for determination of cytokines. LDH was measured by a standard biochemical kinetic UV method and protein was quantified by a colorimetric method (Wiener, Rosario, Argentina). IL-8 (Opt-EIA rabbit IL-8 set; Pharmingen, San Diego, CA, USA), VEGF and TGF-b1 (R&D Systems, Minneapolis, MN, USA) were measured by ELISA according to the manufacturers’ instructions. Pleural vascular permeability was evaluated in 12 rabbits. Two groups of six animals received intrapleural injections of talc or silver nitrate, preceded or not by intravenous bevacizumab (three animals in each group). Three days later, the rabbits received 20 mL of Evans’ blue dye (2.75 mg/mL, USP Pharmacy) intravenously and were sacrificed 1 h later. Pleural fluid was carefully aspirated and the dye concentrations were determined by evaluation of absorbance at 620 nm.
Statistical analysis Results are presented as mean ⫾ SEM. Comparisons between the groups were performed using t-tests (SigmaStat 3.5, Systat Software Inc., Chicago, IL, USA) and a P value < 0.05 was considered significant.
RESULTS Pleural fluid The intravenous administration of bevacizumab reduced the volume of pleural fluid after 3 and 4 days in the talc group and after 3, 4 and 7 days in the silver nitrate group (Fig. 1a). The effusions were exudates, with no significant differences in LDH and total protein levels between treated and non-treated groups (Fig. 1b,c). Intrapleural administration of talc or silver nitrate resulted in significant increases in VEGF levels. As expected, systemic pretreatment with bevacizumab was effective in neutralizing VEGF (Fig. 1d). Both sclerosants induced early increases in IL-8 levels (1 day), followed by progressive decreases over time, with IL-8 levels being lower in animals treated with bevacizumab (Fig. 1e). Injection with the anti-VEGF mAb did not influence the production of TGF-b1, with no significant differences between the groups (Fig. 1f). Although the number of nucleated cells in pleural fluid was higher in the group receiving intrapleural talc, the prior administration of anti-VEGF mAb did not influence this (Fig. 2a). Respirology (2009) 14, 1188–1193
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Figure 1 Effects of bevacizumab on pleural fluid volume and LDH, total protein, vascular endothelial growth factor (VEGF), IL-8 and transforming growth factor-b1 (TGF-b1) concentrations. Bars indicate the values at each time point (1, 2, 3, 4 and 7 days) for the non-treated group (intrapleural talc or silver nitrate only, lighter bar) and the treated group (intrapleural talc or silver nitrate plus intravenous bevacizumab, darker bar) for each variable: (a) pleural fluid volume, (b) LDH, (c) total protein, (d) VEGF, (e) IL-8 and (f) TGF-b1. Data are mean ⫾ SEM, *P < 0.05.
Pleural vascular leakiness was assessed by intravenous injection of Evans’ blue dye. Rabbits treated with bevacizumab showed significantly less leakage of the tracer into pleural fluid, both in the silver nitrate and talc groups (Fig. 3).
Macroscopic examination The administration of anti-VEGF mAb before the profibrotic agents resulted in significant reductions in the pleural adhesions score after 3, 4 and 7 days in both groups (Fig. 2b). None of the animals showed signs of empyema or haemothorax. No pleural adhesions were observed in the left hemithorax.
Microscopic examination Discrete inflammatory alterations were observed in the visceral pleura during the first 7 days after induction of pleurodesis, and these were not influenced by the administration of bevacizumab (Fig. 2c). Finally, pleural fibrosis was temporally progressive, with a fibrosis score higher than 2.5 around the seventh day in the groups treated with talc or silver nitrate, irrespective of the use of anti-VEGF antibody (Fig. 2d). Respirology (2009) 14, 1188–1193
DISCUSSION Over the past few years the effects of VEGF have been investigated in a variety of lung and pleural diseases. Changes in VEGF levels were associated with clinical manifestations in several diseases, including asthma, COPD, OSA, tuberculosis, lung cancer and IPF.16 However, the role of VEGF in the pathophysiology of pleural fluid production is still not completely understood. Studies evaluating VEGF concentrations in serum and pleural fluid of patients with pleural effusions of different aetiologies have demonstrated higher levels of this growth factor in malignant exudates.17–19 The expression of VEGF in pleural exudates is known to increase vascular permeability and, together with other cytokines, to cause fluid accumulation. These findings have encouraged experimental studies to evaluate blockade of VEGF with mAb in order to influence the production of pleural or peritoneal fluid. Yano et al.20 demonstrated that inhibition of phosphorylation of VEGF receptors reduced the volume of malignant effusions in rats with pulmonary adenocarcinoma. Mesiano et al.21 used neutralizing antibodies to block the action of VEGF and showed that the production of ascitic fluid induced by intraperitoneal inoculation of ovarian cancer cells was almost © 2009 The Authors Journal compilation © 2009 Asian Pacific Society of Respirology
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Figure 2 Effects of bevacizumab on total pleural fluid cells, macroscopic pleural adhesions, microscopic pleural Inflammation and fibrosis. Bars indicate the values at each time point (1, 2, 3, 4 and 7 days) for the non-treated group (intrapleural talc or silver nitrate only, lighter bar) and the treated group (intrapleural talc or nitrate plus intravenous bevacizumab, darker bar) for each variable: (a) pleural fluid total cell count, (b) macroscopic adhesion score, (c) microscopic inflammation score and (d) microscopic fibrosis score. Data are mean ⫾ SEM, *P < 0.05.
completely inhibited. There was also an increase in animal survival and a reduction in tumour mass. There are less data regarding benign pleural effusions. However, a recent study evaluating cytokine levels after repeated thoracentesis showed that the increases in pro-inflammatory cytokines and VEGF were positively correlated with the presence of pleural fibrin observed on chest ultrasound.22 Bevacizumab, an mAb with a long T1/2, recognizes all isoforms of VEGF.23 After favourable outcomes in the treatment of advanced colon cancer, bevaci-
zumab was the first angiogenesis inhibitor approved for the treatment of cancer.24,25 Recently, this antibody was also successfully used for the treatment of recurrent pleural effusions in a patient with amyloidosis.26 Experimental studies in the acute phase of pleural inflammation after injection of talc or silver nitrate have demonstrated that these agents induce an inflammatory response characterized by the production of exudative fluid with increased serum and pleural fluid levels of LDH, IL-8 and VEGF. These
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that the use of this agent should be avoided in patients requiring pleurodesis. On the other hand, use of bevacizumab was associated with a reduction in pleural fluid volume, suggesting that VEGF blockade could be a promising target in the treatment of pleural effusions. In conclusion, this study showed that bevacizumab interferes in the acute phase of inflammation induced by talc or silver nitrate, reducing the production of pleural fluid, pleural vascular permeability, IL-8 pleural fluid levels and macroscopic adhesion scores. These data highlight the importance of VEGF in pleural fluid production and suggest that anti-VEGF agents should be avoided in patients requiring pleurodesis.
ACKNOWLEDGEMENTS
0 Talc
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Figure 3 Effect of bevacizumab on pleural vascular permeability. Bars indicate the Evans’ blue dye concentrations in pleural fluid of the 12 rabbits that received intrapleural injections of talc or silver nitrate alone (lighter bar) or preceded by intravenous injections of bevacizumab (darker bar), with sacrifice after 3 days. One hour prior to sacrifice, the rabbits received an intravenous injection of 55 mg of Evans’ blue dye, and dye concentrations were subsequently measured in pleural fluid. Data are mean ⫾ SEM, with P values as indicated.
changes were more pronounced with intrapleural injection of silver nitrate.6,7 In the present study, the volume of pleural fluid was significantly reduced after administration of antiVEGF antibody. This treatment did not change the biochemical characteristics of the pleural fluid (protein and LDH), but reduced the levels of IL-8 and VEGF. Macroscopic analysis of the pleural cavities of animals treated with bevacizumab showed a marked reduction of macroscopic pleural adhesions. Since infiltration of inflammatory cells into the pleural fluid was discrete and similar between treated and nontreated groups, we hypothesized that the marked reduction in fluid volume was mainly due to the effect of VEGF blockade on vascular permeability, rather than its effect on cell recruitment. The injection of Evans’ blue dye in a subgroup of twelve rabbits, using the technique described by Stathopoulos et al.27 confirmed this hypothesis. This study has some limitations. It was an experimental study, performed in animals with normal pleural cavities. However, previous results obtained using a similar model were later validated in clinical studies.3,5 In this context, the clinical application of these findings is ambivalent. Although the efficacy of pleurodesis could not be fully evaluated in this model, the results showed significantly fewer adhesions in animals pretreated with bevacizumab, corroborating the findings from previous studies,28,29 and suggesting Respirology (2009) 14, 1188–1193
Financial support was received from the Foundation to Support Research of the State of São Paulo (FAPESP) and the National Council of Research (CNPq), Brazil.
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