ARTICLE IN PRESS

Respiratory Medicine (2007) 101, 223229

Vascular endothelial growth factor levels in

post-CABG pleural effusions are associated with
pleural inflammation and permeability
Ioannis Kalomenidisa,, Georgios T. Stathopoulosb, Randal Barnetteb,
Spyros Papirisc, Timothy S. Blackwellb, Charis Roussosa, Richard W. Lightb
a

Department of Critical Care and Pulmonary Services, Athens Medical School, Evangelismos Hospital,
45-47 Ipsilandou Street, 10675 Athens, Greece
b
Department of Allergy Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, TN, USA
c
2nd Department of Pulmonary Medicine, Athens Medical School, Attico Hospital, Athens, Greece
Received 9 February 2006; accepted 25 May 2006

KEYWORDS
Post-coronary artery
by-pass grafting;
Pleural
inflammation;
Pleural effusion;
Vascular endothelial
growth factor

Summary
Background: Vascular endothelial growth factor (VEGF) participates in the
pathogenesis of exudative pleural effusions (PEs). In the present study, we
determined the pleural fluid (PF) and serum VEGF levels in patients with postcoronary artery by-pass grafting (post-CABG) PEs.
Methods: Thirty-eight patients with post-CABG (two with bilateral) PEs were
studied. PEs were divided into early (occurring earlier than 30 days after surgery)
and late ones. VEGF levels were measured using ELISA.
Results: (i) Serum and PF VEGF levels did not differ significantly when all the
patients (P 0.053) or those with late effusions (P 0.6) were analyzed; serum
VEGF levels were significantly elevated in comparison to PF VEGF levels in patients
with early (P 0.007) effusions. (ii) Serum VEGF levels were significantly higher in
patients with early than in those with late effusions (P 0.033), while PF VEGF
levels were not significantly different between the two groups (P 0.77). (iii) PF
VEGF levels were higher than corresponding serum levels in 4/24 patients with early
and in 10/16 patients with late post-CABG PEs (P 0.006). (iv) In PEs VEGF levels
significantly correlated with red blood cells (P 0.015), nucleated cells (P 0.003),
protein levels (P 0.002) and lactate dehydrogenase (LDH) levels (P 0.04).
Conclusion: In post-CABG PEs, preferential local production of VEGF in the pleural
cavity is most commonly observed a month or later after surgery. The fact that in PEs

Corresponding author. Tel.: +30210 7201952; fax: +30210 7216573.

E-mail address: ikalom@med.uoa.gr (I. Kalomenidis).

0954-6111/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rmed.2006.05.018

ARTICLE IN PRESS
224

I. Kalomenidis et al.
VEGF levels correlate with markers of pleural inflammation (inflammatory cells and
LDH levels) and vascular hyperpermeability (protein levels) suggests that VEGF may
be involved in the pathogenesis of post-CABG PEs.
& 2006 Elsevier Ltd. All rights reserved.

Introduction
Exudative pleural effusions (PEs) occur in 4289% of
patients subjected to coronary artery by-pass
grafting (CABG).1 The majority of the post-CABG
PEs are small, asymptomatic and resolve spontaneously.2 However, in some patients they are large
enough to cause dyspnea and persist for months,
hence requiring repeated therapeutic thoracenteses.1 Though very rare, pleural fibrosis and
trapped lung may even develop.1 Sadikot and coworkers3 have previously observed that the pleural
fluid (PF) features of PEs occurring within the first
month after CABG differed substantially from those
of PEs occurring later and they speculate that
early effusions are secondary to surgical pleural
trauma and bleeding in the pleural cavity while
late effusions may be immune-mediated. However, the pathogenesis of post-CABG PEs remains
largely unknown.
Accumulating evidence suggests that vascular
endothelial growth factor (VEGF), a major angiogenic cytokine, promotes increased PF production
and accordingly PF accumulation in inflammatory
and malignant pleural diseases.4 This action of
VEGF is mainly attributed to the fact that the
growth factor is a potent enhancer of vascular
permeability which represents the underlying
pathology for fluid exudation in the pleural cavity.4
In this regard, previous studies have found higher
PF VEGF levels in pleural exudates than in
transudates510 and an association between PF
VEGF and markers of pleural vascular hyperpermeability and pleural inflammation, such as PF protein
level, PF/serum protein ratio, PF LDH levels, PF/
serum LDH ratio, nucleated cell count5,10 and PF
neutrophil count.9 It is thus reasonable to hypothesize that VEGF participates in the pathogenesis of
post-CABG PEs.
In the present study, we determined PF and
serum VEGF concentrations in patients with postCABG PEs and we examined whether the presence
of VEGF in the pleural cavity is associated with PF
features indicating pleural inflammation and enhanced pleural vascular permeability. We hypothesized that PF VEGF levels would be significantly
higher than corresponding serum levels in both
early and late effusions and they would correlate

significantly with PF total protein levels, PF lactate

dehydrogenase (LDH) levels and PF nucleated cell
counts.

Patientsmethods
The study was approved by the Institutional Review
Board of the Saint Thomas Hospital, Nashville, TN
and every patient signed an informed consent. We
studied 38 patients who had had CABG within the
previous months and who underwent thoracentesis
for a PE at the St. Thomas Hospital between
December 2002 and September 2003. These patients were included in a previous study by our
group.11 Thirty-eight patients, 36 with unilateral
and 2 bilateral PEs were included in the study (40
effusions in total). Thoracentesis was performed 19
(range: 4159) days after CABG. An effusion was
defined as early when the time elapsed between
surgery and thoracentesis was less than 30 days and
late when this time period was at least 30 days.3
Among the 40 PEs, 24 were early and 16 were
late. One patient with bilateral PEs had one
early and one late effusions. PF and blood
samples were obtained at the same time and
centrifuged at 2000 rpm for 10 min. The supernatants and the serum samples were stored at
80 1C until the assays were performed. PF red and
nucleated cells were counted by manual microscopy. The differential nucleated cell counts in PF
were determined by manually counting 100 cells on
a modified WrightGiemsa-stained smear after cells
had been concentrated by cytocentrifugation at
2000 rpm for 10 min. VEGF level was measured by
ELISA using Quantikine human VEGF ELISA kit (R&D
Systems Inc., Minneapolis, MN, USA). The minimum
detectable level is 5 pg/mL.
Statistics: Values were reported as median
(interquartile range, IQR) since they were not
normally distributed. To assess differences between median values, the MannWhitney or the
Wilcoxon signed rank tests were used, as appropriate. To assess differences between categorical
parameters, the Exact Fishers test was used. To
assess the correlation between two variables the
Spearmans test was used. For statistical analysis

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VEGF in post-CABG pleural effusions

225

and construction of figures the SPSS 11.0 statistical

software (SPSS Inc., Chicago, IL) was used.

Results
PF VEGF levels [228 (103612) pg/mL] did not differ
significantly from the serum levels of the growth
factor but tended to be lower [443 (285808) pg/
mL] (P 0.053). Similarly, there was no significant
difference between PF and serum VEGF when
patients with late effusions were analyzed separately (P 0.6, Table 1). On the contrary, in
patients with early effusions serum VEGF levels
were significantly higher than the PF VEGF levels
(P 0.007, Table 1). In this connection, serum
VEGF levels were significantly higher in patients
with early than in those with late effusions
(P 0.033), while PF VEGF levels did not differ
significantly (P 0.77) between the two groups
(Table 1).
The PF VEGF levels tended to be relatively higher
in the late effusions as compared to the serum
levels than in the early effusions. PF VEGF levels
were higher than the corresponding serum levels in
only 4 of 24 patients with early but in 10 of 16
patients with late post-CABG PEs (P 0.006,
Table 2), indicating that preferential production
of the growth factor in the pleural cavity is
significantly more common a month or later after
surgery. There was a significant negative correlation between the number of days elapsed between
surgery and thoracenteses and serum VEGF levels
(r 0.32, P 0.045) or the difference between
Table 1

serum and PF VEGF levels (r 0.42, P 0.007).

The number of days elapsed between surgery and
thoracenteses did not correlate significantly with
PF VEGF levels.
The levels of VEGF in the PF were significantly
correlated with several PF measurements. A significant correlation between PF VEGF levels and the
following PF features was observed: red blood cell
counts (r 0.384, P 0.015), nucleated cell
counts (r 0.463, P 0.003), number of neutrophils (r 0.345, P 0.032) and lymphocytes
(r 0.458, P 0.003), total protein levels
(r 0.48, P 0.002) and LDH levels (r 0.34,
P 0.004) (Figs. 16). There was not a statistically
significant correlation between PF and serum levels
of VEGF in all patients (P 0.9), in those with early
(P 0.47) or in those with late PEs (P 0.57).

Discussion
In the present study, we determined PF and serum
concentrations of VEGF in patients with post-CABG
PEs. Our main findings were: (i) PF and serum VEGF
levels did not differ significantly when patients
were examined as a whole and in patients with late
PEs, while serum VEGF levels were significantly
elevated compared to PF VEGF levels in those with
early PEs. (ii) Serum VEGF levels were significantly
higher in patients with early than in those with late
effusions, while PF VEGF levels did not differ
significantly between the two groups. (iii) Preferential local production of VEGF in the pleural
cavity, manifested by higher concentration of the

Serum and pleural fluid levels in early and late post-CABG pleural effusions.

Post-CABG PEs

Serum VEGF (pg/mL)

PF VEGF (pg/mL)

P

Early
Late
P

619 (311855)
320 (218597)
0.033

202 (119568)
286 (91883)
0.77

0.007
0.6

 Difference between serum and PF VEGF levels.

 Difference between early and late effusions.

Table 2 Post-CABG PEs with higher or lower VEGF levels than the corresponding serum levels, occurring early
and late after the operation (P 0.006).
Post-CABG PEs

Serum VEGF4PF VEGF

Serum VEGFoPF VEGF

Early

Late

Total

20
4
24

6
10
16

26
14
40

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I. Kalomenidis et al.

Figure 1 The correlation between pleural fluid red blood

cell counts and pleural fluid VEGF levels. Open circles
represent patients with effusions occurring within the
first month after the operation (early) while dark squares
represent those with pleural effusions occurring later
(late).

Figure 3 The correlation between the number of pleural

fluid neutrophils and pleural fluid VEGF levels. Open
circles represent patients with effusions occurring within
the first month after the operation (early) while dark
squares represent those with pleural effusions occurring
later (late).

Figure 2 The correlation between pleural fluid nucleated cell counts and pleural fluid VEGF levels. Open
circles represent patients with effusions occurring within
the first month after the operation (early) while dark
squares represent those with pleural effusions occurring
later (late).

Figure 4 The correlation between the number of pleural

fluid lymphocytes and pleural fluid VEGF levels. Open
circles represent patients with effusions occurring within
the first month after the operation (early) while dark
squares represent those with pleural effusions occurring
later (late).

cytokine in PF than in serum of a patient was

significantly more common in patients with late
than in those with early effusion. (iv) The time

elapsed from surgery inversely correlated with

serum VEGF levels and with the difference between
serum and PF VEGF levels. (v) In PF, VEGF levels

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VEGF in post-CABG pleural effusions

Figure 5 The correlation between pleural fluid total

protein levels and pleural fluid VEGF levels. Open circles
represent patients with effusions occurring within the
first month after the operation (early) while dark squares
represent those with pleural effusions occurring later
(late).

Figure 6 The correlation between pleural fluid LDH and

pleural fluid VEGF levels. Open circles represent patients
with effusions occurring within the first month after the
operation (early) while dark squares represent those with
pleural effusions occurring later (late).

correlated significantly with red blood cell, nucleated cell, neutrophils and lymphocyte counts, as
well as total protein and LDH.

227
Enhanced permeability of the pleural vasculature
typically accompanies pleural inflammation and
both are essential for the pathogenesis of exudative PEs.12 VEGF, an angiogenic growth factor that
induces vascular hyperpermeability and promotes
transmigration of leukocytes into sites of inflammation, is thought to play an important role in the
pathogenesis of pleural exudates.4 This notion is
largely based on the findings of higher PF VEGF
levels in exudative than in transudative PEs510 and
an association between PF VEGF and PF features
indicating pleural vascular hyperpermeability and
inflammation.5,9,10 The PF and serum levels of VEGF
in patients with post-CABG PEs have not been
thoroughly examined before. There are two previous studies by our group that have reported
measurements of PF VEGF in post CABG PEs. The
first of these studies showed that PF VEGF levels in
post-CABG PEs were significantly higher than those
observed in transudative PEs due to heart failure
but significantly lower than those observed in
malignant PEs5 but these findings were not confirmed by the second study.13 In neither of these
studies5,13 were the serum levels of VEGF measured. The present study shows that VEGF levels in
post-CABG PEs correlate significantly with markers
of pleural inflammation (PF nucleated cells, neutrophils and lymphocytes and PF LDH), as well as PF
protein, a marker of pleural vascular permeability.
These observations agree with previous studies
which showed that PF VEGF levels correlate with
PF protein and LDH levels and PF nucleated cell and
neutrophil count5,9,10 in PEs of various etiologies
and imply that VEGF participates in the pathogenesis of post-CABG PEs by promoting pleural inflammation and PF exudation. Nevertheless, It should
be acknowledged that our data are merely suggestive of and do not definitively prove a role for VEGF
in the formation of post-CABG PEs. Interestingly,
although VEGF is implicated in the pathogenesis of
exudative PEs in general,4 direct evidence for such
a role exists only for malignant effusions. In this
regards, in vivo inhibition of VEGF activity suppressed PF accumulation in animal models of
malignant PE.1416 The lack of in vivo studies that
would clarify whether VEGF is involved in the
pathogenesis of exudative PEs of benign etiology
can be attributed to the shortage of relevant
animal models that closely mimic human disease.
The biological basis of the correlation between
PF VEGF levels and RBC counts found in both the
present study and in another study of ours10 on
patients with PEs of different etiologies is unclear.
An association between VEGF and the presence of
blood in the pleural cavity has also been reported
by Ishimoto et al.17 who found that the PF VEGF

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228
levels are significantly higher in hemorrhagic
malignant PEs than in non-hemorrhagic ones. These
observations agree with the findings of an in vivo
study in which the administration of VEGF neutralizing antibodies in mice with malignant ascites not
only suppressed peritoneal microvascular permeability and fluid accumulation but it also reduced
the number of RBC in the peritoneal cavity.18 Since,
neo-vessels formed in the presence of excess
amounts of VEGF are leaky,19 we speculate that
the association between VEGF and the presence of
blood in the pleural cavity most likely reflects the
presence of VEGF-associated angiogenesis.
In patients with post-CABG PEs, the presence of
VEGF in the pleural cavity may be explained by
either intrapleural production from mesothelial or
inflammatory cells4 or diffusion from systemic
circulation. We thus compared PF to serum levels
of the cytokine in order to examine whether
preferential local production of VEGF in the pleural
space occurs in these patients. When patients were
examined as a whole, we observed that serum VEGF
tended to be higher compared to corresponding
serum VEGF but the difference did not reach
statistic significance. When patients with early or
late PEs were examined separately, it became clear
that VEGF was elevated in the blood of patients
with early PE only. In keeping with this, preferential production of VEGF in the pleural space was
significantly more common in patients with late
effusions. These findings should be mainly attributed to the presence of elevated peripheral blood
VEGF levels in the early post-operative period
which may reflect increased VEGF production at
sites of extra-pleural neo-vascularization (surgical
wounds, re-perfused myocardium). The following
are in favor of this assumption: (a) peripheral blood
VEGF levels were significantly higher in patients
with early than in those with late effusions; on the
other hand, although the median level of VEGF in
late effusions was higher than in the early ones, the
difference was not significant. (b) Serum VEGF
levels and the difference between the serum and
the PF VEGF levels declined with time elapsed from
surgery; (c) PF VEGF levels did not correlate with
time from surgery. The above imply that in contrast
to what is happening in extra-pleural sites, the rate
of VEGF production in the pleural cavity does not
change substantially with time and that the
presence of higher serum than PF VEGF concentration in patients with early PEs does not preclude
local production of the growth factor in the pleural
cavity. This interpretation of our results is further
supported by the fact that PF VEGF levels do not
correlate with corresponding serum levels either in
early or in late disease, indicating that the

I. Kalomenidis et al.
presence of the growth factor in the pleural cavity
is not merely the result of diffusion from plasma.
Previous studies have not definitively elucidated
the issue whether preferential local production of
VEGF in the pleural space, manifested by higher PF
than serum levels of the cytokine, characterizes
exudative PE of any etiology. Thus, although such a
finding has been constantly reported in malignant610,20,21 and parapneumonic PEs or empyema,710,20,21 the relationship between PF and serum
VEGF levels in tuberculous PEs is controversial:
three studies showed that VEGF is elevated in PF,79
while other authors reported no difference between PF and serum.6,10,22 Since VEGF inhibition
may be useful in clinical practice to halt PF
accumulation,23 resolving the issue of whether
VEGF or any other hyperpermeability factor involved in the pathogenesis of pleural exudates is
produced mainly in the pleural cavity may be taken
into account to decide whether an inhibitor should
be administered systemically or intrapleurally.
In conclusion, among patients with post-CABG
PE, PFVEGF levels are higher than serum VEGF
levels in the majority of those with PE occurring
after the first month post-operatively suggesting
that VEGF is produced in the pleural space. In
contrast, PF VEGF levels are lower than serum
levels in PEs that occur within the first month. This
is most likely due to extra-pleural production of the
growth factor during the first month post-surgery.
Notwithstanding the source of VEGF, the fact that
its PF levels correlate with markers of pleural
inflammation and pleural vascular hyperpermeability indicates that the growth factor might be
involved in the pathogenesis of post-CABG PEs.
Further studies are required to examine whether
VEGF blockage can be clinically beneficial in
patients with persistent, symptomatic post-CABG
PEs.

Acknowledgments
This study was supported in part by Saint Thomas
Foundation, Nashville, TN, USA. GTS was supported by a scholarship by the Greek State Scholarship Foundation (I.K.Y.).

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