 Induces Vascular
Endothelial Growth Factor Elaboration from Pleural
Mesothelial Cells in Vivo and in Vitro
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Vascular endothelial growth factor (VEGF) increases vascular permeability and is important in pleural effusion formation. In studies
using transforming growth factor 
(TGF-) to produce pleurodesis, we observed that although TGF- was more effective than talc
or doxycycline, it induced transient production of large pleural effusions. We hypothesized that TGF- stimulates VEGF production
in pleural tissues in vivo, and by mesothelial cells in vitro. New
Zealand White rabbits (n = 33) were given TGF-2 (1.7 or 5.0 µg), talc (400 mg/kg), doxycycline (10 mg/kg), or buffer intrapleurally. Pleural fluid was collected at 24 h. Intrapleural injection
of TGF-2 induced a dose-dependent increase in VEGF production.
The pleural fluid VEGF level was 2-fold higher in rabbits given
5.0 µg of TGF-2 than in those given 1.7 µg of TGF-2 and 3-fold
higher than in those given buffer. VEGF levels were higher after
the injection of TGF-2 than after administration of talc or doxycycline. The pleural fluid VEGF correlated significantly with the volume of pleural effusions (r = 0.79, p < 0.00001). In vitro, TGF-2
stimulated a dose-dependent increase in VEGF production from
murine pleural mesothelial cells. At 4 and 24 h, TGF-2, but not
talc or doxycycline, induced a significant increase in VEGF, when
compared with controls. The mesothelial cell VEGF production
was significantly reduced by anti-TGF- antibody in the TGF-2,
talc, and control (buffer and medium) groups. In conclusion, mesothelial cells are an important source of VEGF. TGF- increases
the VEGF production by mesothelial cells in vivo and in vitro.
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Keywords: doxycycline; pleural effusion; talc; transforming growth
factor ; vascular endothelial growth factor
Pleural effusion is common in clinical practice and affects more than 3,000 people per million population each year (1). Pleural fluid accumulates as a result of increased pleural fluid formation and/or reduced drainage (2). The pathophysiology of increased pleural fluid formation remains poorly understood.
Current evidence suggests that vascular endothelial growth factor (VEGF) plays an important role in effusion formation (3). VEGF is a cytokine known for its potent ability to induce vascular leakage, and hence formation of effusion and ascites. It has been shown that tumor cells implanted in the pleural (4) or peritoneal cavity (5) in mice secrete VEGF, which increases the permeability of microvessels and results in development of pleural effusion and ascites, respectively. Conversely, blockade of VEGF receptor phosphorylation inhibited the formation of malignant pleural effusions from lung adenocarcinoma in a murine model (6). In humans, pleural fluid VEGF levels were significantly higher in exudates than in transudates, and VEGF receptors were present at high density in the pleura (7, 8), suggesting that VEGF plays a functional role in the development of effusions. However, the factors responsible for the accumulation of VEGF in the pleural space remain largely unknown.
We investigated the effectiveness of transforming growth
factor (TGF-), a profibrotic cytokine, in producing pleurodesis (9). Interestingly, although TGF- stimulated more
effective pleurodesis than talc or doxycycline, the intrapleural
administration of TGF- induced a large volume of pleural
fluid in the first few days (9). There is evidence that TGF-
can stimulate VEGF release in some epithelial and cancer cell
lines (12), although its effect on mesothelial cells has not
been studied. Also, we have shown that the levels of both
TGF-1 and -2 concentrations correlated with the VEGF levels in all common types of pleural effusions in humans (15).
Hence, we hypothesized that TGF- induces VEGF pleural fluid in vivo and in vitro. The aim of this study is (1) to
compare VEGF production in the pleural space in vivo after
intrapleural injection of TGF- as compared with other profibrotic agents, and (2) to assess the effect of TGF- on VEGF
production of pleural mesothelial cells in vitro.
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For further detail, see online data supplement.
Reagents
TGF-2 and its buffer were obtained from Genzyme (Framingham,
MA). Doxycycline (Fujisawa, Deerfield, IL) and sterilized asbestos-free talc (Sigma, St. Louis, MO) were diluted with 0.9% NaCl for
the animal studies and with Dulbecco's modified Eagle's medium
(DMEM) for cell culture experiments. A murine monoclonal anti-TGF- antibody (1D11) was used to bind the active forms of TGF-1,
-2, and -3 (16).
In Vivo Experiments
Intrapleural administration of agents in rabbitsChest tubes were inserted
into the right pleural cavities of 33 New Zealand White rabbits (1.5-
2.0 kg), using a method previously described (9, 10). TGF-2 at 5.0 µg
(n = 9 rabbits) or 1.7 µg (n = 9), talc at 400 mg/kg (n = 9), doxycycline at 10 mg/kg (n = 3), or TGF-2 buffer (n = 3) in 2.5 ml was injected intrapleurally via the chest tube. The chest tube was aspirated
after 24 h. Pleural fluid was collected on ice, and the supernatant was
stored immediately at 
70° C. The volume and the biochemical analysis of the pleural fluid in these rabbits have been included in previous
reports (9, 10, 17).
To compare the systemic and pleural fluid VEGF levels, plasma
(at baseline and 24 h) and pleural fluid (at 24 h) were collected after
intrapleural TGF-2 (5.0 µg) administration in another five rabbits.
In Vitro Experiments
Culture of mesothelial cellsPleural mesothelial cells were obtained from C57BL/6 mice according to the methodology described for rabbits (18). The cells were cultured in 75-cm2 flasks with DMEM, L-glu-tamine, penicillin-streptomycin, and 10% fetal bovine serum (FBS) at 37° C and 5% CO2. The medium was changed the following day, and thereafter every 3-5 d. Cells of Passage 2 were used for experiments. The cells demonstrated typical cobblestone morphology of mesothelial cells and > 96% were cytokeratin positive. Cell culture materials were purchased from Life Technologies (Grand Island, NY) unless otherwise stated.
Pilot study to establish the toxic and optimal doses for VEGF production.TGF-2 (0.01-100 ng/cm2), talc (1-100 µg/cm2), doxycycline (0.1-
1,000 µg/cm2) in half-log increments, the TGF- buffer, and saline were
applied to mesothelial cells on six-well plates (n = 2 for each dose) for
4, 8, and 24 h. The culture media were then aspirated for VEGF quantification. Cell viability was tested by trypan blue exclusion and the number of cells in each well was measured with a hemacytometer.
Full study.TGF-2 (0.1 ng/cm2), talc (10 µg/cm2), doxycycline (3 µg/
cm2), buffer, or medium alone was applied to mesothelial cells plated in
24-well plates. Doses were determined from the pilot study. Before the
study, the medium was changed to serum-free DMEM to remove any
TGF- in the FBS. The supernatant was collected at 4 h, and stored at
70° C. The cells in each well were then lysed with 500 µl of a lysis
agent containing 0.5% sodium dodecyl sulfate (SDS). The protein in
the lysate was measured by a bicinchoninic acid (BCA) assay (Pierce
Chemical, Rockford, IL), and represented the amount of cells in each
well. Cytokine measurements were normalized to the protein concentration to ensure that the differences were not due to variation of cell
numbers in individual wells. In the in vitro study, VEGF levels were expressed as the fold increase over the medium control group.
The experiment was performed similarly for 8-h and 24-h time points. The full study was repeated for a total of 8 wells (for the 4- and 8-h studies) and 10 wells (for the 24-h studies) for each agent at each time point.
Dose-response of TGF-2-induced VEGF production.To establish
the dose response of VEGF production from TGF-2, mesothelial cells
(five wells in each group) were stimulated with TGF-2 at 0.04, 0.1, and 0.4 ng/cm2 for 24 h and the supernatant was collected for VEGF
measurement. The cell experiment was performed in the same manner as in the full study.
Anti-TGF- antibody study.TGF-2, talc, doxycycline, buffer, and
medium alone were each applied to 10 wells of mesothelial cells, using
the same protocol and doses as described above. Anti-TGF- antibodies (47 µg/ml) were added to half the wells of each group 5 min before the different reagents were applied. The supernatants were collected at 24 h for cytokine measurements.
To ensure that the inhibitory effect seen was not a result of nonspecific inhibition, mouse gammaglobulin (Sigma) was used as a control
antibody for comparison. Mesothelial cells were treated with TGF-2,
TGF-2 with anti-TGF- antibodies (47 µg/ml), TGF-2 with mouse
IgG (47 µg/ml), medium with mouse IgG, and medium only (five wells
in each group) for 24 h, and VEGF was measured in the supernatant.
Cytokine Measurements
Cytokine concentrations were determined with enzyme-linked immunosorbent assay kits (R&D, Minneapolis, MN) and normalized to
protein. In the case of TGF-, all samples were acidified to convert all
TGF- present to the immunoreactive form for measurement, according to the manufacturer's instructions.
Statistics
Data are expressed as means ± standard error. The differences
among groups were compared by one-way ANOVA (parametric) or one-way ANOVA on-ranks (nonparametric). Multiple comparisons
were performed by the Tukey (pairwise comparison) or Dunnett (versus control) method. The Student t test was used to compare the
VEGF levels with or without anti-TGF- antibodies. The Pearson
correlation was used for linear regression analysis. A value of p < 0.05 was considered significant. Data were analyzed with the SigmaStat version 2.03 program (Jandel Scientific, San Rafael, CA).
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In Vivo VEGF Production
Intrapleural injection of TGF-2 induced a dose-dependent increase in pleural fluid VEGF concentrations (Figure 1A). The
pleural fluid VEGF level was significantly higher in rabbits that
received 5.0 µg of TGF-2 than in those given 1.7 µg, which in
turn had higher pleural fluid VEGF levels than rabbits receiving
buffer alone (3,195 ± 275 versus 1,512 ± 129 versus 1,135 ± 215 pg/ml, respectively), p < 0.05. The pleural fluid VEGF level induced after intrapleural injection of 5.0 µg of TGF-2 (3,195 ± 275 pg/ml) was also significantly higher than the VEGF levels after the injection of talc at 400 mg/kg (748 ± 98 pg/ml), and doxycycline at 10 mg/kg (1,127 ± 77 pg/ml) (Figure 1A).
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To establish whether the VEGF was produced locally within
the pleural space or was a result of filtration from the systemic circulation, plasma (at baseline and at 24 h) and pleural fluid (at 24 h) were collected after intrapleural injection of 5.0 µg of
TGF-2 in another five rabbits. There was no difference in the
plasma VEGF levels at baseline and at 24 h (37.2 ± 1.6 and 38.0 ± 2.1 pg/ml, respectively) after intrapleural TGF-2 injections. Pleural fluid VEGF (2,795 ± 272 pg/ml) was about 70-fold higher than the plasma VEGF level at either baseline or
24 h (p < 0.01 for both comparisons) (Figure 1B).
The higher VEGF production induced by TGF-2 relative to
other agents paralleled the development of a significantly larger amount of pleural fluid induced 24 h after the injection of
TGF-2 (Figure 2). The pleural fluid VEGF concentration was
strongly correlated with the volume of pleural fluid induced after
the injection of different agents (r = 0.79, p < 0.000001) (Figure 3). The pleural fluid VEGF levels correlated inversely with lactate dehydrogenase (LDH) or leukocyte concentrations in the effusion (p < 0.00001 for both). There was no significant correlation between the pleural fluid VEGF concentration and the
effusion protein levels (Table 1).
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In Vitro VEGF Production
To assess the ability of mesothelial cells to produce VEGF,
primary cultures of murine pleural mesothelial cells were harvested. The cells were stimulated with TGF-2 and compared
with the other profibrotic agents. The pilot study demonstrated that pleural mesothelial cells were capable of producing VEGF. The lethal doses of these agents on the pleural mesothelial cells are shown in Figures 4A-4C. Interestingly, even
at extremely high doses of TGF-2, none of the cells took up
the trypan blue dye. However, at concentrations > 1 ng/cm2, the
cells demonstrated obvious morphologic changes with severe frag-mentation and in most cells only a residual nucleus was
seen. This morphologic change corresponded with a reduction
in VEGF protein production, confirming cell injury. Cells cultured with 0.9% saline, TGF- buffer, or culture medium
alone all had 
95% survival at 24 h.
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On the basis of the results of the survival studies, the following doses were chosen for each reagent: TGF-2, 0.1 ng/cm2;
talc, 10 µg/cm2; doxycycline, 3 µg/cm2 for the full study of
VEGF production. In general, the optimal dose was about 1-log
dose lower than the lethal concentration. TGF-2 stimulated
significantly higher VEGF production from pleural mesothelial cells in vitro than did talc, doxycycline, or the controls (buffer
or culture medium only) as early as 4 h (Figure 5A). The levels of VEGF at each group increased over time, but the same
pattern was observed at 8 and 24 h (Figure 5B), with the
TGF-2 group inducing the highest amount of VEGF. Results of the preliminary study and the two full studies were highly consistent.
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To further confirm the effect of TGF-2 on VEGF production, a dose-response study was performed by applying TGF-2
at half log increments (0.04, 0.1, 0.4, 1, and 4 ng/cm2) to murine
pleural mesothelial cells. The concentration of VEGF in the
supernatant increased in a dose-dependent manner (Figure 6).
At concentrations higher than 0.4 ng/cm2, VEGF production
reached a plateau, and then decreased with further increases
in TGF-2 concentrations (not shown), mirroring the results
observed in the survival study.
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The concentration of TGF-2 was below the detectable
range (50 pg/ml) in all groups at all time points, including the
group that received TGF-2 (0.1 ng/cm2 or 2 ng/ml) at 4 and 24 h.
TGF-1 levels were measurable in all groups, but did not differ significantly at 4 h. At 24 h, a significantly higher TGF-1
level was present in the TGF-2 group when compared with
the talc or doxycycline group, p < 0.05 (Table 2).
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VEGF production from the mesothelial cells was significantly reduced when TGF-2 was administered with anti-TGF- antibodies, but not with murine gammaglobulin (Figure 7). The VEGF concentrations were reduced in all groups
with the coapplication of anti-TGF- antibody (Figure 8).
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The present study demonstrated that direct intrapleural administration of TGF-2 induced the accumulation of VEGF in
pleural fluids in a dose-dependent fashion in vivo. This increase in VEGF production was associated with the development of large pleural effusions. This study also demonstrated
that pleural mesothelial cells are capable of producing VEGF,
and mesothelial cell production of VEGF increases in a dose-dependent fashion after stimulation by TGF-2. To our knowledge, this is the first study to demonstrate the in vivo induction
of VEGF by TGF-, and the first to show that mesothelial
cells plays a role in VEGF production in the pleural space.
Pleural effusion is a common clinical problem and affects 0.32% of the general population each year (1). Pleural effusion develops when the rate of pleural fluid formation exceeds its rate of absorption (2). Evidence suggests that VEGF plays a critical role in the formation of pleural effusion and ascites (3, 19). VEGF is a potent inducer of vascular hyperpermeability, and can also stimulate vasodilatation and induce fenestrations in endothelial cells in vitro and in vivo (3). These properties contribute to a leakage of plasma and proteins from the vascular space. In animal studies, tumor cells implanted by intrathoracic (4) or intraperitoneal (5) injections produced VEGF and resulted in fluid accumulation. Deletion of the VEGF gene in tumor cells before implantation significantly reduced its expression and effusion formation (4). Likewise, inhibition of VEGF receptor phosphorylation decreased the formation of malignant pleural effusion in mice (6). In humans, VEGF was present in significantly higher concentrations in exudative effusions. Also, VEGF (Fms-like tyrosine kinase 1) receptors are present at high densities in human pleural mesothelial cells, implying an active biologic role for VEGF in the pleural space (7).
Little, however, is known about the factors that induce
VEGF production or the principal source of VEGF in the
pleural space. The present study showed that TGF- can stimulate VEGF accumulation in the pleura, which is associated
with the development of large pleural effusions in rabbits.
TGF- is a ubiquitous multifunctional cytokine with potent
profibrotic activities (20) and, given intrapleurally, could induce more effective pleurodesis than talc and doxycycline (9).
However, we previously observed that whereas TGF- induces more pleural fibrosis, initially it is associated with the
production of larger amounts of pleural fluids compared with
talc or doxycycline (9). TGF- itself does not affect vascular
permeability (21). The present study shows that TGF-2 stimulates significantly more VEGF production than does talc or doxycycline, which can explain our previous observation.
It is interesting that whereas TGF- stimulated the accumulation of VEGF and large pleural effusions, it was also effective in inducing pleurodesis. TGF- is a potent profibrotic agent and we have shown that its intrapleural administration can stimulate significant pleural adhesions even within 24 h (10). By Day 4, most rabbits have developed partial symphysis between the visceral and parietal pleurae. We believe that this rapid induction
of pleural fibrosis allows early obliteration of the pleural space that inhibits continual accumulation of pleural fluids.
What is the source of the pleural fluid VEGF induced after
intrapleural injections of TGF-? The accumulation of VEGF
in the pleural or peritoneal space is believed to be a result of
local production rather than passive diffusion from the systemic circulation (22, 23). This is further supported by the observation that in humans the VEGF levels in malignant effusions were up to 10 times higher than in the corresponding
serum. In our study, the VEGF concentration in the pleural
fluids was about 70-fold higher than the corresponding plasma
VEGF levels, strongly suggesting that the large amount of
VEGF in the effusion originated predominantly from the pleural
space. Previous investigations, however, have focused on the
role of infiltrating cells, such as malignant cells and inflammatory cells (5), as the source of VEGF in the pleural fluids. Our
study demonstrated that resident pleural mesothelial cells express VEGF basally and, more importantly, that this expression is upregulated by TGF-. In humans, the pleural space is
lined by an extensive monolayer of mesothelial cells with an
estimated area of 2,000 cm2 (24). Although infiltrating cells
(such as monocytes [5] and macrophages [25]) may contribute
to the production of VEGF, we believe that the mesothelial
cells represent an important source of VEGF in the pleura.
In this study, we used primary cultures of murine pleural
mesothelial cells and showed that they can be harvested and
cultured to a high degree of purity. Most studies of murine
mesothelial cells employed peritoneal mesothelial cells (26-
28), but it is unknown whether results with peritoneal mesothelial cells can be extrapolated to pleural mesothelial cells.
We also established the toxic doses for TGF-, talc, and doxycycline in mesothelial cells. The lethal dose of talc to murine
mesothelial cells was similar to the published data for human
pleural mesothelial cells and mesothelioma cells (29), suggesting that the behavior of murine pleural mesothelial cells is
similar to that of human mesothelial cells.
This is the first study to show VEGF production from mesothelial cells. A significant rise in VEGF was seen by 4 h after
application of TGF-2, and the VEGF levels in the TGF-2
group remained higher than in the other groups at 24 h. This is
consistent with the results of Chua and coworkers (30) who
demonstrated that in osteoblasts, TGF-1 stimulates an increase in VEGF mRNA expression within 1 h, which peaks
by 2 h. Likewise, TGF--induced VEGF mRNA expression
peaked at 4-8 h and subsided to background levels afterward in AKR-2B (fibroblast) and A549 (lung adenocarcinoma) cell
lines (31).
In humans, there are three isoforms of TGF-: 1, 2, and
3. Although occasional functional differences have been reported, it is generally believed that TGF-1 and -2 have similar biologic functions (32, 33). TGF-1 have been shown to induce VEGF in cultured osteoblasts (30), fibroblasts (34), and
tumor cell lines (13, 14). In the only other study using TGF-2, it was shown to exert the same effect as TGF-1 on the induction of VEGF production from human glioma cells (13). In
our previous study of human effusions, TGF-1 and -2 were
both correlated with VEGF levels (15). Hence, we believe
that the effect of TGF-1 on mesothelial VEGF production
would be similar to what we have shown with TGF-2.
Although we have shown that the administration of TGF-
to mesothelial cells increased VEGF concentrations, the exact
mechanism of induction and the intracellular signaling pathways will require further investigations. On the basis of the effect of TGF- on other cell lines, we speculate that TGF- upregulates VEGF gene transcription (30, 31), although other
mechanisms that can lead to an increase in VEGF levels, such
as release of intracellular stores, cannot be excluded. Also,
VEGF consists of several isoforms with biologic differences
(35). The differential expression of these isoforms by mesothelial cells after stimulation by TGF- warrants further studies.
The TGF-2 (2,000 pg/ml) applied to pleural mesothelial
cells was cleared rapidly. By 4 h, the TGF-2 levels were below the detectable range of 50 pg/ml. In vivo, TGF- is known
to have an extremely short half-life of < 5 min, and is removed
by binding to 
2-macroglobulins and by hepatic clearance
(33). On the other hand, in mesothelial cells the application of
TGF-2 stimulated the production of TGF-1, which increased
with time. This would be in keeping with the previously described autoinduction characteristics
the ability to stimulate
its own productionof TGF- (36, 37).
TGF- exists predominantly in an inactive form through
binding to a latent protein, and the active molecule is released
only on activation (32). The active form is usually present in
minute quantities and this, together with the short half-life,
makes it difficult to measure. Hence, most studies, including
ours, measure the total amount of TGF-. The total amount
of TGF-1 was only slightly higher in the TGF- group, when
compared with other treatment and control groups (Table 2).
Such data are difficult to interpret, as the portion of activated
TGF- in each group is not known. For that reason, we applied an anti-TGF antibody, which binds to all active TGF-
isoforms, but not the latent protein (16), in the cell cultures
and showed that VEGF production was significantly reduced
in all groups. This suggests that the basal VEGF production
from mesothelial cells is in part due to endogenous TGF-.
Factors other than TGF-, such as platelet-derived growth
factor (PDGF), epidermal growth factor (EGF), keratinocyte
growth factor (KGF), interleukin 1 (IL-1), IL-6, and IL-8,
have been shown to induce VEGF production in other cell
lines (3). However, these factors do not consistently induce
VEGF, as does TGF-1. For example, whereas PDGF stimulated VEGF production in vascular smooth muscle cells (38)
and fibroblasts (39), it did not stimulate VEGF production in
human airway epithelial cells. TGF-, but not PDGF, EGF, or
KGF, was capable of inducing VEGF synthesis in transformed
airway epithelial cells (12). Furthermore, TGF- is synergistic
with PDGF (38) and IL-1 (34) in enhancing VEGF production.
We believe the results of this study have clinical implications. Management of recurrent pleural effusions is a common clinical problem. The treatments frequently employed are that of repeated aspiration, or the application of a pleurodesing agent to induce fibrous obliteration of the pleural space. No strategy actually targets the control of increased pleural vascular permeability, which underlies the accumulation of most cases of exudative pleural effusions. Anti-VEGF antibodies have been safely used in a Phase I study (40), and its use against malignant pleural effusions is currently under clinical trial (41). Our results provide further understanding of the mechanism of VEGF induction in the pleural space, and may provide more target options in the pathway of increased pleural fluid formation.
In conclusion, this study showed that TGF- is a potent
stimulator of VEGF production in the pleural space in vivo.
The amount of pleural fluids induced after the intrapleural administration of TGF- correlated significantly with the pleural
fluid VEGF levels. In the in vitro study, we demonstrated that
pleural mesothelial cells are capable of producing VEGF, and
that this production was upregulated by TGF-. Conversely,
anti-TGF- antibodies inhibited VEGF production by mesothelial cells. Targeting the TGF- and VEGF pathway may
provide novel treatment strategies for management of pleural effusions.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Y. C. Gary Lee, M.D., Department of Pulmonary Medicine, St. Thomas Hospital, 4220 Harding Road, Nashville TN 37202. E-mail: ycgarylee{at}hotmail.com
(Received in original form April 2, 2001 and accepted in revised form October 16, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org
Acknowledgments:
The authors thank Genzyme Corporation for providing
the TGF-2, and Dr. Steve Ledbetter for providing the anti-TGF- antibodies used in the experiments.
Supported by the St. Thomas Foundation (Nashville, TN). Dr. Lee is the recipient of a United States-New Zealand Fulbright Graduate Award.
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ABSTRACT
INTRODUCTION
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