Eur Respir J 2007; 29: 761769

DOI: 10.1183/09031936.00061606
CopyrightERS Journals Ltd 2007

Talc mediates angiostasis in malignant

pleural effusions via endostatin induction
N. Najmunnisa*, K.A. Mohammed*, S. Brown#, Y. Su*, P.S. Sriram*, B. Moudgil#,
R. Loddenkemper" and V.B. Antony*

ABSTRACT: Talc remains the most effective sclerosing agent for pleurodesis. However, its
mechanism of action in resolving pleural malignant disease remains unclear.
The present study evaluated the angiogenic balance in the pleural space in patients with
malignant pleural effusions (MPE) following talc insufflation.
Patient pleural fluid samples were collected both before and after talc insufflation. The ability of
pleural mesothelial cells (PMC) and malignant mesothelioma cells (MMC) to produce endostatin
in vitro was compared. The biological effects of pleural fluids and conditioned media from talcactivated PMC on endothelial cells were evaluated by performing proliferation, invasion, tube
formation and apoptosis assays.
Pleural fluids from patients with MPE who received thoracoscopic talc insufflation contained
significantly higher levels of endostatin (median 16.75 ng?mL-1) compared with pre-talc instillation
(1.06 ng?mL-1). Talc-activated PMC released significantly greater amounts of endostatin
(meanSEM 1052.3938.66 pg?mL-1) when compared with a MMC line (134.738.72 pg?mL-1).
In conlusion, talc alters the angiogenic balance in the pleural space from a biologically active
and angiogenic environment to an angiostatic milieu. Functional improvement following talc
poudrage in patients with malignant pleural effusions may, in part, reflect these alterations in the
pleural space.
KEYWORDS: Angiogenesis, malignant pleural effusions, pleura, pleurodesis

alignancy involving the pleura is the

third leading cause of the development
of pleural effusions [1, 2]. Approximately 50% of patients with metastatic cancer
develop malignant pleural effusions (MPE). In
the USA, .150,000 patients with MPE are
diagnosed annually [3]. The presence of a
malignant pleural effusion portends decreased
survival and indicates that the tumour is surgically incurable. The quality of life is greatly
diminished because of symptoms such as dyspnoea and cough [4]. Chemical pleurodesis via
the instillation of a sclerosing agent into the
pleural space is commonly employed in patients
with recurrent MPE [57]. Pleurodesis with talc is
the most commonly used method by chest
physicians to prevent the symptomatic re-accumulation of pleural fluid (PF) in the pleural
cavity of patients [3, 8, 9]. There are several
reasons for choosing talc: it is more effective
when compared with other sclerosing agents [9
11]; it is widely available and cheap; and it is

associated with minimal side-effects [12, 13].

However, recent concerns about adverse events
have tempered its use [14, 15].
When talc is administered into the pleural space, it
interacts with pleural mesothelial cells (PMC)
leading to the development of an inflammatory
response with the release of several cytokines and
chemokines [16, 17]. The interactions between
normal PMC and the tumour cells are largely
unknown. However, the evidence from the present
study suggests that PMC play a key role and may
be critical in modulating the angiogenic environment in the pleural space of patients with MPE.

For editorial comments see page 619.

Endostatin, an inhibitor of angiogenesis, is

released by normal cells and tissues. Endostatin
is a 20-kDa fragment of collagen XVIII that
specifically inhibits endothelial cell migration,
and induces cell cycle arrest, apoptosis, and
reduces tumour growth [18]. The present authors
recently reported that PMC release endostatin and
invading tumour cells enhance the production of
endostatin in PMCs [19]. In the present study, the
authors hypothesise that talc induces PMC to
release the anti-angiogenic factor, endostatin, and

EUROPEAN RESPIRATORY JOURNAL

VOLUME 29 NUMBER 4

AFFILIATIONS
*Division of Pulmonary and Critical
Care Medicine, Dept of Medicine,
College of Medicine, and
#
Dept of Materials Science and
Engineering, College of Engineering,
and Particle Engineering Research
Center, University of Florida,
Gainesville, FL, USA.
"
Lungenklinik Heckeshorn, Berlin,
Germany.
CORRESPONDENCE
V.B. Antony
Division of Pulmonary and Critical
Care Medicine
Dept of Medicine
University of Florida
P.O. Box 100225
Gainesville
FL
USA
Fax: 1 3522714559
E-mail: antonvb@medicine.ufl.edu
Received:
May 08 2006
Accepted after revision:
December 26 2006
SUPPORT STATEMENT
This work was supported in part by
grants NIH RO1 A145338-02 and
NIH RO1 A141877-04 from the
National Institute of Health.
STATEMENT OF INTEREST
None declared.

European Respiratory Journal

Print ISSN 0903-1936
Online ISSN 1399-3003

c
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TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

N. NAJMUNNISA ET AL.

that this may modulate malignant growth on the pleura by

tilting the pro-angiogenic environment of the pleural space to an
anti-angiogenic milieu.
MATERIAL AND METHODS
Cell culture
Human PMC in primary culture (Clonetics Corp., San Diego,
CA, USA) between four and eight passages were used. The
cells were cultured in Hams-199 culture medium (Gibco
Laboratories, Grand Island, NY, USA) containing 10% foetal
bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA,
USA), as reported previously [16]. The malignant mesothelioma cell line (MMC), CRL-2081, was obtained from ATCC
(Rockville, MD, USA) and cultured in RPMI 1640 media with
10% FBS [20]. Primary human umbilical vein endothelial cells
(HUVEC) were obtained from Cell Applications, Inc. (San
Diego, CA, USA). The endothelial cells were cultured according to the manufacturers instructions. In brief, the HUVEC
were grown in growth medium purchased from Cell

a)

Characterisation of talc
Talc (3MgO.4SiO2.H2O; Bryan Corporation, Woburn, MA,
USA) is a tri-layered mineral compound that primarily consists
of pulverised hydrous magnesium silicate. The particle size,
surface area and crystalline impurity data are provided to
enable future comparisons with this study.
Particle size analysis
Talc particle dispersions were prepared in de-ionised water
and measured on a Coulter LS 13320 Particle Size Analyzer
(Beckman-Coulter Inc., Miami, FL, USA), utilising the small
liquid volume module and both laser diffraction and the
polarised intensity differential scattering techniques. The
particle size distributions given in figure 1c were found to be

b)

d)

Counts

20000
15000
10000

200

30
40
50
60
80
100

20

10

3
4
5
6
7

5000

Particle diameter M

FIGURE 1.

30000
25000

6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

Volume %

c)

Applications Inc., in a T-75 flask. The media was changed on

alternate days and the cells were subcultured when HUVEC
reached 6080% confluence. Cells between four and eight
passages were used for all assays.

10

15

20

25

30

20 Cu-Ka

a and b) Scanning electron micrographs of the talc used in the present study deposited on a silicon wafer. Scale bars51 mm (a) and 100 mm (b). c) Particle

size distribution against volume. d) Representative X-ray diffraction pattern for the talc powder used in the current study; the diffraction pattern is the indication of talc peaks.

762

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N. NAJMUNNISA ET AL.

comparable with values obtained with field emission scanning

electron microscopy images (JSM6330F; JEOL Ltd, Tokyo,
Japan).
Surface area analysis
The specific surface area of the talc powder was measured to
be meanSEM 1.900.02 m2?g-1 by the physisorption of
krypton gas (Kr) using the method of BRUNAUER et al. [21]
with a Quantachrome Autosorb 1C-MS apparatus
(Quantochrome Corp., Boyton Beach, FL, USA). Gas adsorption on solid surfaces is commonly used to obtain the specific
surface area and pore size distribution of powdered materials.
Essentially, a dry sample is usually evacuated of all gas and
cooled to a temperature of 77 K, the temperature of liquid
nitrogen. At this temperature, inert gases such as nitrogen,
argon and krypton will physically adsorb on the surface of the
sample. The adsorption process of molecules onto the surface
is measured by monitoring a change in pressure, which is then
used to determine the number of molecules adsorbed and from
this the surface area is determined. Kr adsorption at 77 K is a
much more sensitive technique compared to conventional
methods using nitrogen.
X-ray diffraction analysis
A paste was prepared from talc with a 7:1 mixture of amyl
acetate and colloidan, and then applied to a glass slide. The
sample was air dried prior to measurement. X-ray diffraction
patterns were obtained on a Phillips APD 3700 Powder X-ray
Diffractometer (PANalytical B.V., Almelo, the Netherlands)
with cobalt/nickel-filtered copper-Ka radiation (40 kV,
20 mA).
Talc particle preparation
Talc particles are suspended in endotoxin-free 0.89% normal
saline at a concentration of 10 mg?mL-1. The particles are
washed and then autoclave sterilised. The stock samples of talc
had undetectable levels of endotoxin as determined by limulus
amebocyte lysate assay (Sigma Chemical, St Louis, MO, USA).
Talc samples were dispersed in pH 5.8 Nanopure DI
(Barnstead Thermolyne, Dubuque, IA, USA) water by vortexing. The agitation time and energy was carefully chosen to
ensure maximum de-aggregation and no milling of the talc
particles.
Activation of PMC and MMC with talc
PMC and MMC were treated with varying concentrations of
talc (0, 10, 25, 50, 100 and 200 mg?cm-2) and incubated for 24 h
at 37uC in 5% carbon dioxide and 95% air. The cell culture
supernatants were collected after 24 h. The collected supernatants were liquated and stored at -70uC for further use. The
viability of the PMC was assessed by a Trypan-blue dye
exclusion assay.

TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

age group of the patients included in the present study was

64.212.6 yrs (range 4288 yrs). All patients in the study gave
informed consent, and the study was performed in accordance
with the Lugenklinik Heckenshorn institutional review board
guidelines. Patients in whom malignant pleural effusions were
diagnosed by pleural cytology and who fulfilled the American
Thoracic Society/European Respiratory Society guidelines for the
management of pleural effusions were included for the study.
Medical thoracoscopy was performed under local anaesthesia.
Collection of PF
PF was obtained via thoracentesis, as previously described [22],
from patients with symptomatic malignant pleural effusions
(n516) according to a protocol approved by the institutional
review board. During thoracoscopy, following the removal of
PF, 24 g of sterile, asbestos-free, lipopolysaccharide-free talc
was instilled by insufflation (poudrage) into the pleural cavity
under visual control to ensure homogeneous distribution. On
completion of the procedure, a chest tube was left in place in
all patients. The total amount of PF drainage from the chest
tube in patients who responded to the procedure was
,200 mL following talc insufflation. The chest tube was
removed once the output dropped below 150 cc?24 h-1. PF
was obtained at the beginning of thoracoscopy (baseline),
immediately after thoracoscopy, and at 4- and 24-h postthoracoscopy. All samples were centrifuged and the supernatants were aliquoted into 2-mL samples and frozen at -70uC
until further tests were performed.
Estimation of endostatin by ELISA
Endostatin levels in the PF (0, 4 and 24 h) and culture medium
obtained from activated PMC and MMC (0, 10, 25, 50, 100 and
200 mg?cm-2) and resting PMC and MMC were quantified
using a sandwich enzyme immunoassay kit (Chemicon
International, Inc., Temecula, CA, USA) as previously
described elsewhere [19].
5-bromo-2-deoxyuridine cell proliferation assay
Primary HUVEC were treated with PF obtained from patients
with MPE before and after thoracoscopic talc insufflation and
condition medium (CM) from talc-activated PMC or resting
PMC and incubated for 24 h at 37uC. Cell proliferation was
assessed by a colorimetric assay kit according to the
manufacturers instructions (Calbiochem, San Diego, CA,
USA).

Patient study
A total of 16 patients at the Lugenklinik Heckeshorn (Berlin,
Germany) who had symptomatic MPE and achieved successful
pleurodesis were studied. Pleurodesis was termed successful
when the pleural effusion did not recur at any time during
follow-up until the death of the patient. Of the 16 patients, nine
were female and seven male; seven patients had lung cancer, five
had breast cancer and four had mesothelioma. The meanSEM

Invasion assay
In vitro invasion assays were carried out using the BD Biocoat
Angiogenesis System (BD Biosciences, Bedford, MA, USA)
according to the manufacturers protocols. Briefly, HUVEC
(16105 cells) in suspension were seeded on BD Biocoat
Matrigel (BD Biosciences) 24-well culture plates in the absence
(control) and presence of PF and CM from resting and talcactivated PMC. The lower chamber contained the chemoattractant. After 16 h, the migrated cells were labelled with
calcein acetoxymethyl ester and the fluorescence intensity was
recorded at 450 nm using a fluorescence plate reader
(Cytofluor; Applied biosciences, Gaithersburg, MD, USA).
Data are expressed as a per cent invasion of HUVEC over
control.

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N. NAJMUNNISA ET AL.

Capillary-like tube formation assay

A tube formation assay was performed as previously desribed
[23]. Briefly, a 96-well culture plate was coated with 100 mL of
matrigel per well and allowed to polymerise for 30 min at
37uC. HUVEC at a density of 36104 cells?well-1 were plated in
0.3 mL of serum-free RPMI 1640 media. The cells were
pretreated with PF and CM from talc-activated PMC and
resting PMC for 1 h at 37uC. The cells were placed on matrigel,
after a 10-h incubation, four to six randomly chosen fields (at
106 magnification) from the sample were photographed, and
total tube areas were analysed by the Axio-vision image
programme (Carl Zeiss, Houston, TX, USA).

Statistical methods
Statistical differences between experimental groups were
tested using ANOVA. The KruskalWallis test was used for
analysis of differences between more than two groups and the
MannWhitney U-test was used for analysis between two
specific groups. Data were considered significant if p,0.05.
RESULTS
X-ray diffraction crystallography
In order to provide more detailed information of the talc used
in this study, X-ray diffraction was used to provide the
crystalline fingerprint analysis of talc. A scanning electron
micrograph of talc is shown as figure 1a. Figure 1d represents
a characteristic X-ray diffractrogram from the talc used in the
present study.
PF from patients with MPE contains endostatin
PF endostatin was measured sequentially before and after talc
pleurodesis. The PFs were collected at 0, 4 and 24 h after the
procedure. Endostatin levels were found to be significantly
higher in all MPE collected at 24 h. Following talc insufflation,
the statistical difference among groups was not significant
(p50.194) when comparing lung cancer (median (interquartile
range) 17.55 (11.9621.49) ng?mL-1); breast cancer (15.26
(10.6620.26) ng?mL-1) and malignant mesothelioma (18.42
(15.8120.12) ng?mL-1) patients, with breast cancer (1.05 (0.7
1.50) ng?mL-1) and malignant mesothelioma (1.55 (1.12
2.2) ng?mL-1) patients at 0 h PF (1.5 (1.01.67) ng?mL-1). The
0 h (before insufflation of talc) sample was considered as
control. The data are presented in figure 2 as box plots
showing upper and lower quartile ranges.
764

VOLUME 29 NUMBER 4

FIGURE 2.

Pleural fluid endostatin levels over time. Endostatin levels were

measured in the pleural fluids of lung cancer (h), breast cancer (&) and malignant
mesothelioma (&) patients with malignant pleural effusions prior to (0 h) and post
(4 and 24 h) thoracoscopy. Boxes represent the medianinterquartile range and
bars represent upper and lower quartile ranges. *: p,0.05 versus 0 h; ***:
p,0.001 versus 0 h.

Talc-activated PMC release endostatin

PMC and MMC were treated with various concentrations of
talc (0, 10, 25, 50, 100 and 200 mg?cm-2) and incubated for 24 h
at 37uC. The culture supernatants were collected after 24 h.
PMC activated with talc at a concentration of 25 mg?cm-2
released significantly higher levels of endostatin (meanSEM
1052.3938.66 pg?mL-1; p,0.001) when compared with MMC
(134.738.72 pg?mL-1). MMC produced minimal levels of
endostatin at all concentrations tested (fig. 3). However, with
increasing concentrations of talc the endostatin levels significantly decreased in PMC. These data indicate that at higher
doses, talc has an inhibitory effect on PMC endostatin
production, and thus affects the biological activity of the cells.
1200

***

***

1000

Endostatin pgmL-1

Annexin-V fluoroscein isothiocyanate and propidium iodide

staining
HUVEC at 80% confluence were pretreated with PF and CM
from talc-activated PMC and resting PMC. After 48 h,
detached cells in the medium were collected and the remaining
adherent cells were harvested by trypsinisation. The cells
(16105) were washed with PBS and resuspended in 250 mL of
binding buffer (annexin-V fluoroscein isothiocyanate (FITC)
kit; Becton Dickinson, Franklin Lakes, NJ, USA) containing
10 mL of 20 mg?mL-1 propidium iodide (PI) and 5 mL of
annexin-V FITC. After incubation for 10 min at room temperature in a light-protected area, the samples were analysed
on a FACSCalibur flow cytometer (Becton Dickinson). FITC
and PI emissions were detected in the FL-1 and FL-2 channels,
respectively. Subsequent analysis was carried out using with
CellQuest software (Becton Dickinson).

800

***

600
400

***

200
0

10

25

50

Talc concentration

FIGURE 3.

100

200

gcm-2

Talc-induced endostatin release in pleural mesothelial cells (PMC;

&) and malignant mesothelioma cells (MMC; h). PMC and MMC were activated
with varying concentrations of talc (0200 mg?cm-2) for 24 h and the endostatin
released was measured. Data are expressed as the meanSEM of four independent
experiments. ***: p,0.001 versus MMC.

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N. NAJMUNNISA ET AL.

TABLE 1

TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

The percentage viability of pleural mesothelial

cells exposed to various concentrations of talc
as evaluated by Trypan-blue dye exclusion

Talc mg?cm-2

98.83.8

10

97.64.3

25

97.23.5

50

96.45.2

100

91.87.5

200

87.59.3

Data are presented as the meanSEM of six individual experiments.

In order to evaluate whether higher doses of talc have any

cytotoxic effect on PMC, the PMC viability was estimated with
a Trypan-blue dye exclusion assay on PMC activated with
various concentrations of talc; the data are presented in table 1.
Talc alters the angiogenic activity (as measured by
proliferation, invasion and tube formation of HUVEC) in PF
from patients who receive talc insufflation
The endothelial cells were pretreated with PF obtained from
patients with malignant pleural effusions post-thoracoscopy at
0, 4 and 24 h. The following components of angiogenesis were
evaluated.
1) The proliferative capacity of the cells was determined by 5bromo-2-deoxyuridine (BrdU) cell proliferation assay (fig. 4a).
PF collected 24 h post talc insufflation significantly decreased
the proliferation of HUVEC (42.963.18%) compared with PF
prior to talc insufflation. Culture supernatant obtained from
talc activated PMC significantly inhibited the proliferation
of HUVEC (33.064.64%) compared with PMC-CM

FIGURE 4.

(18.612.64%). Addition of anti-human endostatin antibody

inhibited the anti-proliferative effect and the proliferation of
HUVEC was enhanced significantly (fig. 4b).
2) Endothelial cell invasion and tube formation were evaluated
in matrigel in order to evaluate the biological activity of PF and
CM of talc-activated PMC. The invasion of endothelial cells
was significantly inhibited in the PF samples obtained from
24 h post talc insufflation when compared with the 0 h control
(24.253.85%; p,0.05) against the chemoattractant vascular
endothelial growth factor (VEGF; fig. 5a). There was an
18.842.28% (p,0.05) and 10.491.21% inhibition of invasion
of endothelial cells in the samples treated with culture
supernatants of talc-activated and resting PMC, respectively
(fig. 5b).
3) Tube formation of endothelial cells was significantly
disrupted in samples treated with PF from patients 24 h post
talc insufflation. A meanSEM decrease of 52.586.64% in
tube length formation was noticed compared with the 0 h PF
sample (fig. 6). The tube-like structure formation of endothelial
cells was also disrupted, and a significant decrease in the tube
length was noticed in the samples treated with culture
supernatants of talc-activated (25 mg?cm2) PMC compared
with resting PMC (23.843.64%; p,0.05), suggesting the
release of an anti-angiogenic factor (fig. 7).
PF collected post thoracoscopy and conditioned medium
from talc-activated PMC induces apoptosis in HUVEC
An early step in the process of cell death is the redistribution of
phosphatidylserine (PS) from the inner leaflet to the outer
leaflet of the plasma membrane, due to the loss of membrane
asymmetry [24]. The externalised PS can be visualised by
incubating intact cells with a fluorescent derivative of the
protein annexin-V, a phospholipid-binding protein. PI is a
fluorochrome used to label DNA. Annexin-V FITC staining
was performed in order to determine the apoptosis of HUVEC

Effect of pleural fluid (PF) and pleural mesothelial cell conditioned media (PMC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation.

a) HUVEC proliferation in presence of PF obtained before (0 h) and after (4 and 24 h) thoracoscopy. b) HUVEC proliferation in presence of talc-activated and resting PMCCM. Recombinant endostatin was used as a negative control and vascular endothelial growth factor (VEGF; 20 ng?mL-1) was used as a positive control. Data expressed are
the meanSEM of four independent experiments. *: p,0.05 versus 0 h PF and control; ***: p,0.001 versus 0 h PF and control; #: p,0.05 PMC-CM versus PMC + talc.

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TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

a)

220

***

***

d) 8

120

200
*

Endothelial cell invasion

% inhibition over control

c)

70
b)

b)

170

150

Tube formation mmmm-2

Endothelial cell invasion

% inhibition over control

a)

N. NAJMUNNISA ET AL.

5
4

3
2
1
0

24

Endothelial cell PF treatment h

100

FIGURE 6.

Effect of pleural fluid (PF) on tube formation in human umbilical

vein endothelial cells (HUVEC). A representative image of tube formation in HUVEC

in the presence of PF obtained from patients a) before (0 h) and after b) 4 h and c)

50

24 h talc thoracoscopy. d) Tube formation (mm?mm-2) in HUVEC. Data presented

SFM

VEGF

5% FBS

Chemoattractant

FIGURE 5.

Effect of pleural fluid (PF) and pleural mesothelial cell conditioned

media (PMC-CM) on human umbilical vein endothelial cell (HUVEC) invasion.

a) HUVEC invasion after pre-treatment with pleural fluids obtained before (control,

are the meanSEM of three separate experiments. *: p,0.05 versus 0 h PF.

those who have had successful pleurodesis [4, 20, 24, 25]. The
present study demonstrates that talc induces PMCs to release
the anti-angiogenic factor, endostatin, which may be responsible for containment of tumour growth in the pleural space
and may account, in part, for the improved clinical status of
patients who have had successful pleurodesis.

0 h; h) and after (4 h: &; and 24 h: &) thoracoscopy. b) HUVEC invasion in control

and after pre-treatment with talc-activated (&) and resting (&) PMC-CM. Data are
expressed as % HUVEC invasion over control against chemoattractants. SFM:
serum-free media; VEGF: vascular endothelial growth factor; FBS: foetal bovine
serum. *: p,0.05; ***: p,0.001 versus 0 h PF and control.

induced by PF or culture supernatants obtained from resting

PMC and talc-activated PMC. The apoptosis was noticed in
HUVEC cultured in the PF samples obtained 24 h post talc
insufflation when compared with the 0 h control (meanSEM
31.242.85% versus 7.892.85%; p,0.05). The 4 h PF showed
12.682.27% apoptosis of HUVEC (data not shown).
Approximately 21.862.68% (p,0.05) and 18.682.21%
(p,0.05) apoptosis was observed in the samples treated with
culture supernatants of talc-activated and resting PMC,
respectively. Actinomycin-D was used as a positive control
(fig. 8).
DISCUSSION
Lung, breast and ovarian cancers account for 5065% of MPE.
Life expectancy following the diagnosis of a MPE is usually
,6 months. Talc has been widely used for pleurodesis in
patients with MPE. Talc is known to induce apoptosis in
malignant cells and to improve survival and quality of life in
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VOLUME 29 NUMBER 4

Although talc is by far the most effective sclerosing agent, with

a success rate .90%, its use remains controversial [5, 2628].
The quality of talc, including the particle size and dose used for
pleurodesis, has shown varied effects on the morbidity of the
patients with malignant MPE. The present authors believe that
the magnitude of adverse effects is greater when the particle
size is ,10 mm [12, 29, 30], compared with graded talc with the
smallest particles removed [31]. Moreover, most of the
reported cases of lung injury and acute respiratory distress
syndrome have been associated with a high talc dose [12, 29].
The talc used in the current study was characterised, providing
information on size and crystalline finger print analysis. This
analysis can be important when exposing in vitro cultures of
cells to talc particulates, as well as for studies carried out on
clinical samples from patients treated with talc.
Angiogenesis is critical for tumour growth as well as the
establishment of metastatic deposits. Malignant cells release
several angiogenic factors that promote new blood vessel
formation and tumour growth [3235]. The present authors
have previously demonstrated that cancer cells induce PMC to
release VEGF [32]. However, a normal mesothelium is critical
in maintaining the dynamic balance of angiogenic versus antiangiogenic factors [19, 32]. The present authors model clearly
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N. NAJMUNNISA ET AL.

TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

a)

b)

c)

d)

Tube formation mmmm-2

e) 12

current authors have demonstrated that PF obtained from

patients with MPE after thoracoscopic talc insufflation inhibits
proliferation of endothelial cells. The decrease in proliferation
of HUVEC cells may, in part, be due to the cells undergoing
apoptosis [20]. In the present study, there was 21.863.24%
and 31.244.78% annexin-V FITC binding in the HUVEC pretreated with talc-activated PMC-CM and PF of the patients
after 24 h post thoracoscopy, respectively.

10
8
6

*
*

4
2
0
a

Endothelial cell treatment

FIGURE 7.

Effect of pleural mesothelial cell conditioned media (PMC-CM) on

tube formation in human umbilical vein endothelial cells (HUVEC). A representative

image of tube formation in HUVEC exposed to CM obtained from a) control pleural
mesothelial cells (PMC), b) vascular endothelial growth factor (VEGF; 20 ng?mL-1),
c) PMC + talc (10 mg?cm-2) and d) PMC + talc (25 mg?cm-2). e) Tube formation
measured in mm?mm-2. Data presented are the meanSEM of three separate
experiments. *: p,0.05 versus control PMC.

demonstrate that malignant cells growing on the pleural

surface gain metastatic potential by inducing the production
of VEGF, thus creating a pro-angiogenic milieu in the pleural
space [32].

Tumours depend on an invasive vasculature for their growth.

Endostatin present in the PF of the talc-insufflated patient is
known to modulate this aspect of angiogenesis. To confirm this
effect, capillary tube formation and the invasive capacity of
endothelial cells was evaluated. The ability of endothelial cells
to form a network of tube-like structures on matrigel was
significantly disrupted when the cells were co-cultured in PF
obtained after 24 h of talc insufflation. The conditioned media
from talc-activated PMC also significantly disrupted the tube
formation of endothelial cells. Additionally, inhibition of the
invasion of endothelial cells was also noticed. The decreases in
tube formation and invasion of endothelial cells could be
attributed to the talc-induced production of the anti-angiogenic
factor, endostatin. The disruption of tube-length formation of
endothelial cells may be due to a decrease in cell number and
apoptosis of endothelial cells.
Several reports in the literature suggest that patients who have
had successful pleurodesis have improved clinical status and
outcomes compared with patients with failure of pleurodesis
[4, 24, 25, 38]; some patients may live longer than 1 yr. The
presence of high levels of endostatin in the post-thoracoscopy
PF and the in vitro data from the present study suggest that the
inhibition of angiogenesis in the pleural space may contribute
to eventual outcomes in these patients. Angiogenic factors are
produced by malignant cells, and VEGF is one of the best
described pro-angiogenic factors responsible for the angiogenic switch. The control of the angiogenic switch relies upon
the balance between pro- and anti-angiogenic factors [39, 40].
The current study demonstrates that an angiogenic environment is present in the pleural space in MPE. The amount of
endostatin present in talc-untreated MPE is insufficient to tilt
the balance. The addition of talc results in an increase in the
amount of endostatin released by normal PMC, with a
resultant shift in the balance to angiostasis.

Angiogenesis is composed of several components, including

increases in proliferation of endothelial cells and invasion of
the surrounding tissue by new blood vessels [3335, 37]. The

Talc is a cheap, safe and effective sclerosant for MPE [3, 9]. The
present authors previously reported that talc induces apoptosis
of malignant cells in the pleural space [20]. The present study
clearly demonstrates a previously unknown property of talc,
i.e. its ability to stimulate normal PMC to release endostatin.
Controlling angiogenesis in the pleural space is a logical step
towards the treatment of MPE. Although clinical trials with
endostatin in the treatment of other types of malignancies have
not met with expected results, the current authors believe that
it definitely has an important role in controlling tumour
growth in the pleural space, but do not believe that endostatin
alone will be an answer for the treatment of MPE. However,
drugs that target angiogenesis in the pleural space could
complement traditional chemotherapeutic agents. A multipronged approach, i.e. targeting tumour cells with chemotherapeutic agents, inhibition of angiogenic factors with anti-VEGF

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RUIZ et al. [36] reported that angiogenic activators were higher

in neoplastic pleural effusions than nonmalignant effusions.
However, no significant difference in endostatin levels was
noticed. The present study demonstrates that PF obtained from
patients with MPE who undergo thoracoscopic talc insufflation
contain significantly higher levels of endostatin when compared with PFs from patients who have not received
intrapleural talc. Lower levels of endostatin in PF before talc
insufflation is consistent with the current authors hypothesis
that the PF in MPE is predominantly pro-angiogenic. Talc
insufflation appears to cause a marked shift in the pleural
milieu from angiogenic to angiostatic.

767

TALC AND PLEURAL MESOTHELIAL CELL ENDOSTATIN

a) 200

N. NAJMUNNISA ET AL.

b)

c)

Cell counts

160
120
*

80
40
0

d) 200

e)

f)

160
Cell counts

120
80
40
0

100

FIGURE 8.

101
102
103
Relative fluorescence intensity

104 100

101
102
103
104
Relative fluorescence intensity

100

101
102
103
Relative fluorescence intensity

104

Effect of pleural fluid (PF) and pleural mesothelial cell conditioned media (PMC-CM) on annexin-V expression in human umbilical vein endothelial cells

(HUVEC). The HUVEC were cultured either in the presence of a) serum-free media, b) PMC-CM, c) talc-activated (25 mg?cm-2) PMC, d) PF for 0 h, e) PF for 24 h or f)
actinomycin-D. Data presented are representative of three separate experiments. Horizontal bars represent the percentage of apoptosis as follows: a) 2.82, b) 18.68, c) 21.86,
d) 7.89, e) 31.24, and f) 82.96. *: p,0.05 versus SFM; #: p,0.05 versus 0 h PF.

antibodies and the use of anti-angiogenic factors may have

better success.
In conclusion, the findings of the present study support the use
of talc as a sclerosing agent in the treatment of patients with
recurrent malignant pleural effusions. The environment in the
pleural space prior to the administration of talc, as represented
by the pleural fluid from patients with malignant pleural
effusions, is strongly pro-angiogenic. This microenvironment
supports the growth of tumour cells by the presence of
angiogenic factors. The insufflation of talc leads to a dramatic
and immediate change in the pleural space with a reversal of
the angiogenic activity present in the pleural fluid from proangiogenic to angiostatic. The major contributor that moves the
biological balance and tips the scale towards angiostasis
appears to be endostatin.

5
6

8
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