From the Department of Bacteriology and Immunology,
the Haartman Institute, and the Department of Obstetrics and
Gynecology, Haartmaninkatu 2, FIN-00290 University of Helsinki, and
¶ Molecular/Cancer Biology Laboratory, Haartman Institute, POB 21, FIN-00014 University of Helsinki, Helsinki, Finland
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) is a
prime regulator of normal and pathological angiogenesis. Three related
endothelial cell growth factors, VEGF-B, VEGF-C, and VEGF-D were
recently cloned. We have here studied the regulation of VEGF-C, a
lymphatic endothelial growth factor, by angiogenic proinflammatory
cytokines. Interleukin (IL)-1 induced a concentration- and a
time-dependent increase in VEGF-C, but not in VEGF-B,
mRNA steady-state levels in human lung fibroblasts. The increase in
VEGF-C mRNA levels was mainly due to increased transcription rather
than elevated mRNA stability as detected by the nuclear run-on
method and by following mRNA decay in the presence of an inhibitor
of transcription, respectively. In contrast, angiopoietin-1 mRNA,
encoding the ligand for the endothelial-specific Tek/Tie-2 receptor,
was down-regulated by IL-1
. Tumor necrosis factor-
and IL-1
also elevated VEGF-C mRNA steady-state levels, whereas the IL-1
receptor antagonist and dexamethasone inhibited the effect of IL-1
.
Experiments with cycloheximide indicated that the effect of IL-1
was
independent of protein synthesis. Hypoxia, which is an important
inducer of VEGF expression, had no effect on VEGF-B or VEGF-C mRNA
levels. IL-1
and tumor necrosis factor-
also stimulated the
production of VEGF-C protein by the fibroblasts. Cytokines and growth
factors have previously been shown to down-regulate VEGF receptors in vascular endothelial cells. We found that the mRNA for the VEGF- and VEGF-C-binding VEGFR-2 (KDR/Flk-1) was stimulated by IL-1
in
human umbilical vein endothelial cells, whereas the mRNA levels of
VEGFR-1 (Flt-1) and VEGFR-3 (Flt-4) were not altered. Our data suggest
that in addition to VEGF, VEGF-C may also serve as an endothelial
stimulus at sites of cytokine activation. In particular, these results
raise the possibility that certain proinflammatory cytokines regulate
the lymphatic vessels indirectly via VEGF-C.
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INTRODUCTION |
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Angiogenesis, most commonly involving sprouting of capillaries
from preexisting blood vessels, starts with proteolysis of the
subendothelial extracellular matrix followed by migration and
proliferation of endothelial cells (1). Thereafter, the newly grown
endothelium differentiates into tubelike structures, which are
stabilized by mesenchymal components, preventing leakage and fused to
allow blood circulation. The significance of angiogenesis is now widely
accepted in many physiological and pathological conditions (2).
Angiogenesis is needed for embryonic development, for several female
reproductive functions, and for wound healing and other repair
processes, including collateral blood vessel formation in ischemic limb
and heart diseases (1-3). Angiogenesis also occurs in nonmalignant
diseases, such as diabetic retinopathy, atherosclerosis, psoriasis, and
rheumatoid arthritis, and its importance in solid tumor growth has been
demonstrated in multiple experimental models. Several growth factors
and cytokines promote angiogenesis in animal models. Most of them,
including tumor necrosis factor
(TNF)1- and interleukin
(IL)-1, do not stimulate endothelial cell growth in culture and are
thus called indirect angiogenic factors (4). Once tubes composed of
endothelial cells are formed, angiogenesis is apparently completed by
stabilization of these vessels through migration of pericytes and
production of basement membrane components. Both of these processes
have been suggested to be facilitated via endothelial cell specific
Tek/Tie-2 receptor ligand angiopoietin-1 (Ang-1) (5-8). In addition to
angiogenic inducers, several endogenous inhibitors of angiogenesis have
been recently characterized (2, 9). The switch to an angiogenic
phenotype in tumors may thus be triggered by overproduction of factors
in favor of angiogenesis or reduction of inhibitor release, both of
which can depend on the activation of oncogenes and inactivation of
antioncogenes (1).
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a glycoprotein with homology to platelet-derived growth factor (PDGF), and it is translated into multiple protein forms due to alternative exon splicing (reviewed in Refs. 10-14). VEGF is a prototype of directly acting angiogenic factors, because it is secreted in a biologically active form, its receptors are found at sites of angiogenesis, and it is the most specific endothelial cell growth factor known thus far. VEGF also induces vascular permeability, regulates production of proteases and their inhibitors, and promotes endothelial cell differentiation, movement, and survival, which are related to its angiogenic properties.
Up-regulation of VEGF synthesis by hypoxia and by several indirectly acting angiogenic factors also supports its role in angiogenesis. VEGF transduces its signals through Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2) tyrosine kinase receptors. However, mitogenesis of endothelial cells may be signaled exclusively via VEGFR-2. Genetic disruption of VEGF or its receptors has indicated that they are necessary for vasculogenesis (differentiation of endothelial cells from their precursors) and angiogenesis (15-18). Further, injection of VEGF can induce blood vessel formation, whereas antibodies against VEGF, soluble extracellular domain of VEGFR-1 or overexpression of a dominant negative VEGFR-2 inhibit angiogenesis and subsequent tumor growth in animal models (13, 14, 19, 20).
VEGF-B (also known as VEGF-related factor) (21-25), VEGF-C (also known as VEGF-related protein and VEGF-2) (26-30), and c-fos-induced growth factor (also known as VEGF-D) (31, 32) are recently discovered members of the VEGF family. Thus, this family is currently composed of five members, including also the placenta growth factor (33). VEGF-B is an endothelial cell mitogen, which is expressed in most tissues, but especially highly in the heart and skeletal muscle (23). VEGF-C induces endothelial cell migration and vascular permeability (26, 34), but, unlike VEGF, it is a relatively weak mitogen for blood vascular endothelial cells (28, 30). VEGF-C mRNA is expressed at low levels in many tissues, most prominently in the heart, placenta, skeletal muscle, ovary, and the small intestine, and it is also present in certain tumor cell lines (26, 35). VEGF-C is the ligand for VEGFR-2 and for the third member of the VEGF receptor family, VEGFR-3 (also known as Flt-4), which is espressed mainly on venous endothelium in early embryos and lymphatic endothelium in adult tissues (27, 36, 37). Indeed, mice overexpressing VEGF-C in basal keratinocytes developed hyperplastic lymphatic vessels in the skin (38). Although VEGF induces growth of vascular and lymphatic endothelial cells in vitro, it seems to be specific for blood vessel growth in vivo (39).
The existence of a gene family consisting of several related growth factors suggests that these family members have overlapping but distinct functions and that they may be differentially regulated. The lymphatic endothelial growth factor VEGF-C (38, 39) in particular could be important in inflammatory conditions by controlling the composition and pressure of interstitial fluid and by facilitating lymphocyte trafficking. We have therefore studied the regulation of the expression of VEGF-C in comparison with VEGF and VEGF-B by the angiogenic proinflammatory cytokines and anti-inflammatory agents.
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MATERIALS AND METHODS |
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Cell Culture and Cytokine and Inhibitor Treatments--
Diploid
human lung fibroblasts (IMR-90, American Type Tissue Collection
CCL-186) were grown in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal calf serum (FCS), glutamine, and antibiotics
(Life Technologies, Inc.). Prior to initiation of the experiments, the
cells were maintained in 0.5% FCS for 48 h. Cells were then
incubated with human rIL-1 (0.001-10 ng/ml), rIL-1
(10 ng/ml),
or rTNF-
(50 ng/ml) in 0.5% FCS for the time periods indicated. All
cytokines and growth factors were obtained from R&D Systems (Abingdon,
UK). Dexamethasone (1-10 µM), indomethacin (10 µM), cycloheximide (10 µg/ml), and phorbol 12-myristate
13-acetate (PMA) (10 ng/ml) were from Sigma. Dexamethasone (10 µM) was either added simultaneously with IL-1
(10 ng/ml) for 6 h, or alternatively, 1 µM of
dexamethasone was first added to the starvation medium for 12 h,
and incubation was then continued with dexamethasone and with or
without 1 ng/ml IL-1
for 6 h. Human rIL-1 receptor antagonist
(IL-1ra; 200 ng/ml) was used to inhibit the effect of IL-1
(0.2 ng/ml); in some experiments, human rPDGF-BB (100 ng/ml) served as a
positive control. Human umbilical vein endothelial cells (HUVECs) were
isolated according to the standard protocol (40), cultured on
gelatin-coated dishes in medium 199 (Life Technologies, Inc.)
containing 10% FCS, endothelial cell growth supplement (50 µg/ml;
Upstate Biotechnology Inc., Lake Placid, NY), heparin (5 units/ml,
Sigma), antibiotics, and glutamine, and the experiments were performed
at passage 4. Before initiation of the experiment, HUVECs were grown
confluent, then starved in 5% FCS in medium 199 for 24 h, after
which either IL-1
(10 ng/ml) or TNF-
(50 ng/ml) were added to
the starvation media for 6 h.
Hypoxia Treatment-- Confluent cultures of IMR-90 cells were grown on 10-cm diameter tissue culture plates containing 10 ml of DMEM and 0.5% FCS. These cultures were exposed to hypoxia by incubating the cells for 6 h in the Anaerocult A anaerobic culture jar supplied by Merck (Darmstadt, Germany).
Isolation and Analysis of RNA--
Total RNA was isolated using
the Trizol reagent (Life Technologies) and quantitated by absorbance at
260 nm. The poly(A)+ mRNA fraction was isolated with
the Poly(A)Ttrack mRNA isolation system (Promega, Madison, WI). For
Northern blots, 20 µg of total RNA or 1 µg of poly(A)+
mRNA were denatured in 1 M glyoxal, 50% dimethyl
sulfoxide, and 10 mM phosphate buffer at 50 °C for 60 min, electrophoresed, and transferred to nylon membranes (Micron
Separations Inc., Westborough, MA), which were baked for 1 h at
80 °C and then UV-irradiated for 6 min (Reprostar II UV, Camag,
Muttenz, Switzerland). Hybridization was performed with a mixture of
[-32P]dCTP (NEN Life Science Products)-labeled
fragments of human VEGF cDNA (a 583-base pair EcoRI
fragment), VEGF-B167 cDNA (nucleotides 1-382,
GenBankTM accession no. U48801) and VEGF-C cDNA
(nucleotides 495-1661, GenBankTM accession no. X94216)
with the Prime-a-gene kit (Promega). A glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probe served as a control for the equal gel
loading and cyclooxygenase-2 as a positive control for a
cytokine-induced gene (41). The human Ang-1 (5) cDNA probe was a
1.4-kb reverse phase-polymerase chain reaction fragment containing the
open reading frame, a kind gift from Dr. Yuji Gunji. The receptor
probes were the following: VEGFR-1 (nucleotides 706-2310;
GenBankTM accession no. X51602), VEGFR-2 (nucleotides
6-715; GenBankTM accession no. X61656), VEGFR-3
(nucletotides 1-595; GenBankTM accession no. X68203),
Tie-1 (nucleotides 1-2190; GenBankTM accession no.
X60957), and Tek/Tie-2 (nucleotides 1550-2285; GenBankTM
accession no. L06139, a kind gift from Eola Kukk). Probes were purified
by gel filtration through nick columns (Pharmacia, Uppsala, Sweden) and
used at 1 × 106 cpm/ml. Hybridizations were done at
42 °C for 16 h in solution containing 50% formamide, 6× SSC,
0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin,
100 mg/ml herring sperm DNA, 100 mg/ml yeast RNA, and 0.5% SDS.
Membranes were washed three times at 50 °C for 15 min in 0.1× SSC
and 0.1% SDS. The probed blots were exposed to Fuji medical x-ray film
after quantitation of the signals with the Fujifilm IP reader
Bio-Imaging analyzer BAS1500 (Tokyo, Japan) and the MacBas software
supplied by the manufacturer and visualized by autoradiography.
Analysis of Transcription--
For nuclear run-on analysis
IMR-90 cells were first starved for 48 h and then incubated for
3 h with or without IL-1 (10 ng/ml) in DMEM supplemented with
0.5% FCS (10 15-cm dishes each). Total RNA was extracted from one
control dish and one IL-1
-treated dish and used for Northern
blotting. The nuclei were isolated from nine control and nine
IL-1
-treated dishes as described by Greenberg and Ziff (42).
Extraction of the RNA and nuclear transcription were performed as
described by Celano et al. (43). [32P]UTP
(3000 Ci/mmol)-labeled nuclear RNA samples (1 × 107
cpm/sample) were then hybridized to linearized plasmid DNA (pcDNA I) containing inserts of VEGF-C and GAPDH, or no insert. The probed blots were quantitated with the Bio-Imaging Analyser using the MacBas
software, after which background was subtracted from VEGF-C and GAPDH
signals.
Analysis of mRNA Half-life--
Degradation of the mRNAs
were studied in human IMR-90 lung fibroblasts. The cells were starved
in DMEM supplemented with 0.5% FCS for 48 h and shifted to medium
containing 5% FCS for 4 h prior to mRNA stability
measurements. After two washes with DMEM, medium containing 0.5% FCS
and actinomycin D (10 µg/ml; Sigma) as an inhibitor of transcription,
with or without IL-1 (10 ng/ml), was added for 1, 2, or 4 h.
Detection of VEGF-C Protein--
Confluent IMR-90 cell cultures
were first starved for 48 h in DMEM supplemented with 0.5% FCS
and then preincubated with or without IL-1 (10 ng/ml) or TNF-
(50 ng/ml) for 4 h. After two washes with phosphate-buffered saline,
metabolic labeling was carried out by addition of 100 µCi/ml of
Pro-mix L-35S in vitro cell labeling
mix (Amersham, Buckinghamshire, UK) to methionine- and cysteine-free
Eagle's minimal essential medium, with or without the cytokines. After
6 h the media were collected and centrifuged to remove cellular
debris. Aliquots of the culture media were precipitated with
trichloroacetic acid and measured for incorporated radioactivity. Equal
counts/min of the media were immunoprecipitated with anti-VEGF
antiserum as described earlier (34). The immunoprecipitated proteins
were separated in SDS-polyacrylamide gel electrophoresis and visualized
and quantitated by the Bio-Imaging analyzer system.
Statistical Analysis-- Statistical significance was calculated by Student's t test. All results are shown as means ± S.E., and p < 0.05 was selected as the statistically significant value.
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RESULTS |
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Kinetics and Concentration Dependence of IL-1-induced VEGF-C
mRNA Expression--
To study the kinetics and
concentration-dependence of VEGF-C mRNA regulation, IMR-90 cells
were stimulated with 10 ng/ml IL-1
for 2-48 h or with 0.001-10
ng/ml for 6 h. As shown in Fig.
1A, the induction was already
evident at the earliest 2-h time point, maximum induction was reached
by 4 h, and the mRNA levels began a steady decline after an
incubation period of 6 h, reaching base line around the 12-h time
point. Results with different concentrations of IL-1
are shown in
Fig. 1B, indicating that the maximal effect was obtained by
1 ng/ml.
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Proinflammatory Cytokines Induce VEGF-C mRNA
Expression--
Expression of VEGF-C transcripts was studied in
serum-starved human IMR-90 fibroblasts. The cells were exposed to
IL-1, IL-1
, or TNF-
for 6 h, after which total RNA was
isolated, electrophoresed, and subjected to Northern blot hybridization
using a mixture of the VEGF, VEGF-B, and VEGF-C probes. The filters
were also hybridized separately with probes for cyclooxygenase-2,
serving as a known cytokine-responsive gene (41) and for GAPDH, which
served as a loading control. As shown in Fig.
2A, IL-1
, IL-1
, and
TNF-
elevated VEGF-C mRNA steady-state levels. Fig.
2B shows a densitometric evaluation of the VEGF-C mRNA
levels induced by IL-1
from eight independent experiments (a
3.41 ± 0.35-fold increase). IL-1
also stimulated VEGF mRNA
expression (a 2.55 ± 0.55-fold increase), but not that of VEGF-B.
Interestingly, although the steady-state mRNA levels for Ang-1 were
readily detectable under basal conditions, they were down-regulated by
IL-1
(10 ng/ml for 6 h) by 50 ± 9% as calculated from
seven separate experiments (the controls versus IL-1
-treated, p < 0.05).
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Effect of Dexamethasone, Indomethacin, Inhibition of Protein
Synthesis, and IL-1ra on IL-1-induced VEGF-C mRNA
Expression--
Fig. 3 shows the
inhibitory effect of the anti-inflammatory and antiangiogenic
glucocorticoid dexamethasone (4) on IL-1
-induced VEGF and VEGF-C
mRNA expression. Furthermore, while dexamethasone completely
blocked the effect of IL-1
on VEGF mRNA induction, it inhibited
only partially the elevation of VEGF-C mRNA steady-stale levels. In
contrast, indomethacin, which is a nonsteroidal anti-inflammatory drug
and an inhibitor of prostanoid production, did not inhibit the effects
of IL-1
(not shown).
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Effect of Hypoxia on IL-1-induced VEGF-C mRNA
Expression--
Hypoxia is an important stimulus for angiogenesis and
an inducer of VEGF expression (10-14). In IMR-90 cells, hypoxia
induced VEGF mRNA but not VEGF-B or VEGF-C mRNAs (Fig.
5). Further, when hypoxia was combined
with IL-1
treatment, no additional VEGF stimulation was found in
comparison to that of hypoxia alone. This is in contrast with the
stimulation obtained with PDGF, which was potentiated by hypoxia (45)
(Fig. 5). Moreover, cyclooxygenase-2 expression was recently reported
to be induced by hypoxia in human vascular endothelial cells (46), but
this did not seem to be the case in the IMR-90 cells (Fig. 5).
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Effect of IL-1 on VEGF-C mRNA Transcription and
Stability--
To investigate a possible mechanism contributing to
IL-1
-induced VEGF-C mRNA expression, we performed nuclear run-on
experiments, which indicated that transcription of VEGF-C is induced to
similar extent as are the steady state levels of VEGF-C mRNA (Fig.
6A). We also measured the
half-life of VEGF-C mRNA in the presence of an inhibitor of
transcription, actinomycin D, with or without IL-1
for 1-4 h. As
shown in Fig. 6B, the half-life of VEGF-C in the absence of
IL-1
was approximately 3.5 h and it was somewhat prolonged in
the presence of IL-1
(t1/2 > 4 h). VEGF
mRNA was degraded considerably faster with a half-life of
approximately 1 h, whereas the IL-1
-stabilized mRNA had a
half-life of approximately 2 h, which is consistent with the
results obtained in rat aortic smooth muscle cells (47). The level of
VEGF-B mRNA was not reduced during the 4 h experiment. These
data suggest that the mechanism of the IL-1
-induced VEGF-C mRNA
expression operates mostly at the transcriptional level.
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IL-1 and TNF-
Stimulate the Production of the VEGF-C
Protein--
IL-1
and TNF-
stimulated the production of
metabolically labeled VEGF-C protein (6- and 9-fold, respectively), as
detected by immunoprecipitation of the metabolically labeled proteins
secreted to the culture media with the use of VEGF-C-specific
polyclonal antibodies (Fig. 7). The major
protein precipitated from the culture medium corresponds to the
partially processed VEGF-C isoform, which is biologically active
(34).
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Effect of IL-1 on Expression of VEGFs and VEGFRs in Vascular
Endothelial Cells--
Since very little is known about VEGF, VEGF-B,
or VEGF-C expression in vascular endothelial cells, we investigated
their mRNA levels in HUVECs. Both VEGF-B and VEGF-C, but not
VEGF, were detected in HUVECs, and the VEGF-C mRNA level
was increased by treatment of the cells with either IL-1
or TNF-
(Fig. 8A). Furthermore, IL-1
stimulated VEGFR-2 mRNA (Fig. 8B). In agreement
with the earlier reports (48, 49), PMA stimulated VEGFR-1 and VEGFR-2 mRNA levels, whereas TNF-
decreased VEGFR-1 mRNA. Tek/Tie-2
and the related endothelial-specific tyrosine kinase receptor Tie-1 (50) were not affected by treatments with the cytokines or PMA (Fig.
8B).
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DISCUSSION |
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We have here studied the regulation of the expression of three
members of the VEGF family in response to proinflammatory cytokines. All VEGFs studied were expressed in human lung fibroblasts. In serum-starved cells, the levels of VEGF-B and VEGF-C mRNAs were similar, but higher than those of VEGF. IL-1, IL-1
, and TNF-
induced VEGF and VEGF-C mRNAs, but VEGF-B expression was not
affected by these cytokines. In contrast, IL-1
decreased Ang-1
mRNA levels. This is consistent with the hypothesis that the
Tek/Tie-2 signaling pathway may be important in the destabilization of
blood vessels during angiogenesis and play a role later during the
stabilization of the newly formed vessels (5-8). We also found that
IL-1
and TNF-
stimulate the production of a partially processed
form of the VEGF-C protein, which is an active ligand of the lymphatic endothelial cell receptor VEGFR-3 (34). Since the mature form of VEGF-C
protein is released from its precursor as a result of proteolytic
cleavages, production or activation of such proteases could be also
modulated at sites of cytokine activation in vivo. It
remains to be studied whether cytokines regulate these
post-translational processes.
VEGF expression has previously been shown to be stimulated by IL-1
in human synovial fibroblasts (51), by IL-1
in rat aortic smooth
muscle cells (47), and by IL-6 in tumor cell lines (52). In addition to
our fibroblast model, TNF-
was found to stimulate VEGF expression in
keratinocytes (53) and in tumor cell lines (52), but not in vascular
smooth muscle cells (47). Hypoxia induces VEGF expression by
transcriptional activation via hypoxia-inducible factor-1 and by
post-transcriptional stabilization of the mRNA (54-59). The
mechanism of action of IL-1
was also suggested to depend both on
transcriptional activation and on post-transcriptional mRNA
stabilization (47). In the case of VEGF, the rapid mRNA decay
depends on AU-rich protein binding instability elements found in the
3'-untranslated region (55, 58), which are not present in the VEGF-C
mRNA (60). This is consistent with our data, which show that VEGF-C
mRNA has a relatively long half-life and that only a small
stabilizing effect can be obtained with IL-1
. All this suggests that
mRNA stabilization is not as important for VEGF-C regulation as it
is for VEGF. Indeed, our nuclear run-on data suggest that IL-1
induces expression of VEGF-C at the transcriptional level. Li et
al. (47) hypothesised that IL-1
may facilitate transcriptional
activation of the VEGF gene via AP-1 sites found in its 5'-flanking
region (61). Data published by Chilov et al. (60) indicate
that the 5'-flanking region of the VEGF-C gene contains a putative
binding site for NF-
B, which is an important transcription factor
for signal transduction of proinflammatory cytokines (62).
Interestingly, NF-
B sites have also been identified in the mouse,
but not the human, VEGF gene (59, 61).
Glucocorticoids are important anti-inflammatory agents that also
inhibit angiogenesis and reduce brain tumor-associated vascular permeability (63, 64). Heiss et al. (65) recently suggested that the inhibition of vascular permeability by dexamethasone is
delivered via inhibition of VEGF production. Further, serum- and
PDGF-induced, but not hypoxia-induced VEGF expression was inhibited by
dexamethasone in glioma cell lines (65). We found that IL-1-induced
expression of VEGF and VEGF-C was inhibited by dexamethasone in IMR-90
fibroblasts. Whether the effect of dexamethasone is due to direct
inhibition of VEGF-C transcription or due to interference with the
IL-1
signaling pathway remains to be studied. Proinflammatory
cytokines stimulate the production of prostanoids and dexamethasone
inhibits it via the inducible cyclooxygenase-2 pathway (41, 66). Since
prostanoids are considered to be indirect angiogenic agents acting by
inducing synthesis of VEGF (51, 67), we investigated, whether the
induction of VEGF-C by cytokines is prostanoid-dependent.
Indomethacin, a cyclooxygenase-inhibiting nonsteroidal
anti-inflammatory drug, did not inhibit the IL-1
-induced VEGF-C
mRNA expression confirming the mechanism to be independent of
prostanoids.
Namiki et al. (68) recently reported that HUVECs and human
dermal microvascular endothelial cells express VEGF mRNA, but only after induction with PMA or hypoxia. In HUVECs we detected VEGF-B and VEGF-C mRNAs but no VEGF mRNA. Furthermore, VEGF-C mRNA level was increased by treatment with IL-1 and TNF-
.
Since neither VEGF nor VEGF-C expression has been detected in
endothelial cells in vivo, the significance of these
findings remains to be determined. In fact, VEGF-C was detected in
mesenchymal cells around the vessels expressing VEGFR-3, suggesting a
paracrine mode of action (27). It is, however, tempting to speculate
that, in defined conditions, such as during cytokine activation, VEGF-C could modulate endothelial cell responses in an autocrine manner.
VEGFRs are specifically expressed on endothelial cells in
vivo (14). Their expression levels are high during embryogenesis, low in most adult tissues, and again elevated in many tumors. However,
only a few examples of VEGFRs mRNA regulation exist. TNF- was
recently shown to down-regulate VEGFR-1 and VEGFR-2 mRNAs in HUVECs
and in human aortic endothelial cells (48), whereas PMA induced all
three VEGFR mRNAs in HUVECs (49). We found that IL-1
elevated
the level of VEGFR-2 mRNA in HUVECs. These data support the
hypothesis that cytokines could also induce responsiveness of
endothelial cells to VEGF and VEGF-C by increasing the expression of
VEGFR-2.
We have here shown that proinflammatory cytokines up-regulate the expression of VEGF-C. Our data suggest that, in addition to VEGF, VEGF-C may serve as an endothelial stimulus at sites of cytokine activation. Since VEGF-C primarily targets endothelium of lymphatic vessels, these results raise the possibility that certain proinflammatory cytokines affect the lymphatic vessels indirectly via VEGF-C. This may be important in controlling the composition and pressure of interstitial fluid and in facilitating lymphocyte trafficking and thus have an important role in inflammatory processes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ulf Eriksson for expert help with reagents, and Kaija Antila, Ritva Javanainen, and Tapio Tainola for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Finnish Academy, the Finnish Cancer Organizations, and Helsinki University Central Hospital Research Funds.
§ To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Research Laboratory, University of Helsinki, Haartmaninkatu 2, FIN-00290 Helsinki, Finland. Tel.: 358-9-471 4981; Fax: 358-9-471 4801.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by Finska Läkarsällskapet.
1 The abbreviations used are: TNF, tumor necrosis factor; Ang-1, angiopoietin-1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; IL, interleukin; PDGF, platelet-derived growth factor; PMA, phorbol 12-myristate 13-acetate; ra, receptor antagonist; r, recombinant; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
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REFERENCES |
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