KDR activation is crucial for
VEGF165-mediated
Ca2+ mobilization in human
umbilical vein endothelial cells
Sonia A.
Cunningham1,2,
Tuan M.
Tran1,
M. Pia
Arrate1,
Robert
Bjercke1, and
Tommy A.
Brock1
1 Department of Pharmacology,
Texas Biotechnology Corporation, Houston, 77030; and
2 Department of Integrative
Biology, Pharmacology, and Physiology, University of Texas Health
Science Center, Houston, Texas 77225
 |
ABSTRACT |
We have prepared a
polyclonal mouse antibody directed against the first three
immunoglobulin-like domains of the kinase insert domain-containing
receptor (KDR) tyrosine kinase. It possesses the ability to inhibit
binding of the 165-amino acid splice variant of vascular endothelial
cell growth factor (VEGF165) to
recombinant KDR in vitro as well as to reduce
VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first
three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR
antibody is able to completely block
VEGF165-mediated intracellular
Ca2+ mobilization in HUVEC.
Therefore, it appears that binding of VEGF165 to the fms-like tyrosine
kinase (Flt-1) in these cells does not translate into a
Ca2+ response. This is further
exemplified by the lack of response to placental growth factor (PlGF),
an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate
the effects of submaximal concentrations of
VEGF165. Surprisingly, the
VEGF-PlGF heterodimer was also very inefficient at eliciting a
Ca2+ signaling event in HUVEC. We
conclude that KDR activation is crucial for mobilization of
intracellular Ca2+ in HUVEC in
response to VEGF165.
tyrosine kinase; neutralizing antibodies; kinase insert
domain-containing receptor; vascular endothelial cell growth factor
 |
INTRODUCTION |
VASCULAR ENDOTHELIAL CELL growth factor (VEGF), a
mitogen that promotes angiogenesis, vasculogenesis, and endothelial
differentiation, is crucial for embryonic development. This is
exemplified by VEGF-deficient embryos that do not survive beyond
midgestation even in the heterozygous state (6, 13). VEGF binds to and
activates the kinase insert domain-containing receptor/fetal liver
kinase (KDR/Flk-1) and fms-like tyrosine kinase (Flt-1) (30). In
endothelial cells, where both receptors are expressed simultaneously,
it is not clear whether VEGF stimulation results in redundant
signaling. However, unique signaling pathways and/or
expression patterns are apparent, since knockout mice reveal differing
lethal phenotypes for each receptor. Whereas Flk-1 is essential for
embryonic endothelial cell differentiation and vasculogenesis (25),
Flt-1 plays a crucial role in the organization of the developing
vasculature (14).
Several studies concerning VEGF signaling have been performed in
endothelial cells expressing endogenous receptors. Thus VEGF promotes
phosphorylation of phosphatidylinositol 3-kinase, rasGAP, and Nck in
bovine aortic endothelial cells (17). Most recently, VEGF has been
shown to stimulate tyrosine phosphorylation of p125 focal adhesion
kinase and paxillin in human umbilical vein endothelial cells (HUVEC;
Ref. 1). However, it is not possible to delineate the individual
pathways activated by each receptor in these studies.
Our understanding of the separate signaling pathways utilized by
KDR/Flt-1 has mainly come from recombinant expression of these
receptors in heterologous cells. Expression in porcine aortic endothelial cells (PAEC) defines a role for KDR but not Flt-1 in
VEGF-induced cell proliferation (31). This is further supported by the
lack of proliferation obtained by expression and activation of Flt-1 in
NIH/3T3 cells (24). VEGF also appears to be a mitogen for pancreatic
ductal epithelium that expresses the KDR but not the Flt-1 receptor
(21). Other functions attributed to KDR in PAEC are changes in cell
morphology, actin reorganization, membrane ruffling, and chemotaxis
(31). In addition to endothelial cells, recent reports have described
Flt-1 expression in pericytes (18, 28) and monocytes (3, 8). However,
it is not clear whether the signaling mechanisms utilized by Flt-1 in
this background are modified by the expression of specific
intracellular signaling proteins.
One of the first cell-based signaling assays to be analyzed for VEGF
was the mobilization of intracellular
Ca2+ in endothelial cells (16).
Activation of phospholipase C (PLC) results in the hydrolysis of
phosphatidylinositol 4,5-bisphosphate into
D-myo-inositol
1,4,5-trisphosphate
(IP3) and
diacylglycerol. The elevated IP3
levels cause release of Ca2+ from
the endoplasmic reticulum. Both autophosphorylated KDR and Flt-1 are
capable of binding to the src homology 2 (SH2) domains of PLC-
(10,
23), and tyrosine phosphorylation of PLC-
occurs following VEGF
stimulation of cells transfected with Flt-1 (23) and KDR (29). This
would suggest that the Ca2+
mobilization in response to VEGF stimulation of endothelial cells is
due to redundant signaling through both the Flt-1 and KDR receptors. To
test this, we raised neutralizing mouse polyclonal antiserum against
the VEGF binding region of KDR. With this tool, we directly addressed
the involvement of endogenously expressed KDR with respect to
Ca2+ signaling in primary cultures
of endothelial cells.
 |
METHODS |
Preparation of polyclonal mouse serum.
Immunoglobulin domains 1-3 encompassing amino acids 1-338 of
KDR were fused to the carboxy-terminal end of murine IgG2A as previously described for Flt-1 (amino acids 1-348; Ref. 11). The
construct was subcloned into the pBakPak vector (Clontech), and
recombinant baculovirus was prepared according to standard procedures.
Sf21 cells were infected with virus, and the serum-free medium
containing the secreted fusion proteins was harvested after 72 h.
Recombinant protein was purified over protein A Sepharose columns (KDR)
and used for injection into mice. Mice were injected with 50 µg of
antigen diluted 1:1 in PBS and complete Freund's adjuvant on
day
0. Animals were boosted with 25 µg
of antigen diluted 1:1 in PBS and incomplete Freund's adjuvant on
days
21 and
45. The serum used for these
experiments was collected 10 days after each boost. Further boosts and
bleeds were performed as necessary.
Cell culture.
HUVEC (Cascade Biologics) in this study were used between
passages
1 and
5. Cells were grown on gelatin-coated
plates in medium 199, 2 mM
L-glutamine, 0.2 µg/ml heparin
(Sigma), and 100 µg/ml endothelial mitogen (Biomedical Technologies).
125I-labeled 165-amino acid
VEGF-receptor binding on recombinant protein.
Both Flt-1 and KDR-Fc fusions were immobilized on Immulon 4 plates
using a goat anti-mouse capture antibody.
125I-labeled 165-amino acid splice
variant of VEGF (VEGF165) at 4 ng/ml was bound to the receptor fusions at room temperature for 90 min
in binding buffer consisting of medium 199 (BRL), 1% BSA, and 25 mM
HEPES (pH 7.4). Nonspecific counts were estimated using unlabeled
VEGF165 at 400 ng/ml to compete.
For inhibition, various dilutions of antiserum against KDR-Fc were
preincubated with receptor for 60 min before radiolabeled
VEGF165 addition. Wells were
washed three times with cold PBS and directly counted in a gamma counter.
125I-VEGF165-receptor
binding on endothelial cells.
HUVEC were plated on 48-well plates at 10,000 cells/well.
Binding was performed essentially as described for the recombinant protein after 4 days of culture. After the PBS wash, cells were detached from the plate with 150 µl of 0.1 M NaOH and counted in a
gamma counter.
Endothelial
Ca2+
mobilization assay.
HUVEC were detached from monolayers and loaded with 2 µM fura 2-AM
for 30 min at 37°C in PBS. Cells were washed and resuspended at a
final count of 0.5 × 106
cells/ml. For antibody studies, polyclonal serum at dilutions of 1:100
to 1:400 was incubated with cells at 37°C for 30 min before
stimulation with VEGF165.
Fluorescence was monitored at 340 and 380 nm, and intracellular
Ca2+ concentration was estimated
as described previously (16). For each experiment, a baseline resting
fluorescence was measured for 100 s before stimulation with 10 ng/ml
VEGF165, 190 ng/ml placental
growth factor (PlGF; 149 amino acids; R&D), or 100-500 ng/ml
VEGF-PlGF heterodimer.
 |
RESULTS |
From our previous work and that of others focusing on the Flt-1
receptor, we reasoned that the VEGF binding determinants of KDR would
reside in the first three immunoglobulin-like domains (4, 11, 12, 15).
Therefore, we injected a recombinant KDR encompassing this region into
mice as an immunogen to produce polyclonal antiserum.
To determine the neutralizing capacity of the polyclonal antibody, we
performed an in vitro
125I-VEGF165
binding assay against the recombinant receptor. Figure 1 shows that, at a 1:100
dilution and a receptor coating of 50 ng/well, the anti-KDR serum was
able to inhibit VEGF165 binding to
KDR by ~70%. A full inhibition was not achieved, and this was probably due to the high levels of recombinant receptor used in this in
vitro assay. The extent of inhibition was reduced with progressive
dilutions. Thus the polyclonal serum possesses a proportion of
antibodies against regions in the extracellular domain that are
important for ligand binding. Flt-1 and KDR display 33% identity in
the first three immunoglobulin domains as defined by our construct. Because both receptors bind
VEGF165 with high affinity, we
tested for the ability of the anti-KDR serum to cross-neutralize
VEGF165 binding to Flt-1. In
experiments performed in parallel, Flt-1 bound 8,261 ± 1,435 counts/min (cpm) per well, whereas KDR bound 3,315 ± 470 cpm/well (n = 4). Figure 1 shows that
the KDR antibody at dilutions of 1:100 and greater has no effect on
VEGF165 binding to Flt-1 and thus
defines its specificity for KDR.

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Fig. 1.
Specificity of anti-kinase insert domain-containing receptor (anti-KDR)
polyclonal serum: inhibition of
125I-labeled 165-amino acid splice
variant of vascular endothelial cell growth factor
(VEGF165) binding to KDR but not
to fms-like tyrosine kinase (Flt-1) in vitro. Immobilized recombinant
receptors were preincubated with dilutions of anti-KDR or nonimmune
serum at 1:100, 1:300, and 1:900 for 60 min before addition of 4 ng/ml
125I-VEGF165.
Results are expressed as %inhibition of
VEGF165 binding. KDR 1-3 and
Flt 1-3, first 3 immunoglobulin-like domains of KDR and Flt-1,
respectively.
|
|
Having demonstrated the neutralizing capability and specificity of the
polyclonal serum on recombinant protein, we performed similar binding
assays on the surface of HUVEC. All experiments were performed on cells
of low passage number and similar confluencies. Figure
2 depicts the average results from four
experiments; it is clear that the anti-KDR serum inhibits a proportion
of the 125I-VEGF165
binding sites available on the cell surface. Inhibition starts to
plateau at serum dilutions of ~1:100. A maximum inhibition of ~60%
was recorded at 1:50 dilution. Limited serum supply did not allow the use of lower dilutions. Nonimmune serum had
negligible effects. The serum is useful for detection of recombinant
KDR by Western blot analysis in addition to immunocytochemistry.
However, it is unable to detect the low levels of expression of KDR in total cell lysates of HUVEC (data not shown).

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Fig. 2.
Inhibition of
125I-VEGF165
binding to primary cultures of endothelial cells. Human umbilical vein
endothelial cells (HUVEC) were preincubated with dilutions of anti-KDR
or nonimmune serum at 1:50, 1:100, 1:300, and 1:900 for 60 min in
medium 199. 125I-VEGF165
at 4 ng/ml was added. Nonspecific counts were determined with 100-fold
excess of unlabeled VEGF165.
|
|
We and others have shown that PLC-
can bind to autophosphorylated
KDR and Flt-1 receptors (10, 23, 29). Furthermore, we have identified
the phosphotyrosine binding sites on each receptor that are important
for this interaction. A consequence of PLC-
activation is the
mobilization of intracellular
Ca2+. We assessed the ability of
our neutralizing KDR antibody to inhibit
VEGF165-induced
Ca2+ mobilization in HUVEC. A
representative experiment is shown in Fig.
3 (n = 3).
The first trace depicts the response to 10 ng/ml VEGF165. After a short delay, a
rapid transient rise of intracellular Ca2+ concentration, peaking at
~100 nM within 60 s, is observed following VEGF165 addition. Preincubation of
cells with the anti-KDR serum attenuated this rapid
Ca2+ rise at dilutions of 1:100.
At 1:50 dilution of antibody, no further effect was recorded (data not
shown). With further dilutions of the antibody, the inhibition was
gradually lost. Nonimmune serum at similar dilutions was without
effect.

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Fig. 3.
Anti-KDR serum inhibits VEGF-induced intracellular
Ca2+ signaling. HUVEC (~1 × 106) were loaded with
fura 2-AM, and intracellular Ca2+
concentration
([Ca2+]i)
was monitored. VEGF165 (10 ng/ml)
was added at arrow to cells preincubated with or without anti-KDR serum
(KDR ab) at 1:100 and 1:400 dilutions (dil). NMS, nonimmune serum at
1:100. Approximate
[Ca2+]i
was calculated according to Ref. 16.
|
|
Figure 4 shows that acute addition of a
1:100 dilution of the anti-KDR polyclonal serum itself does not
activate the KDR receptor and result in
Ca2+ mobilization. Both normal
mouse serum (Fig. 4A) and anti-KDR serum (Fig. 4B) do, however, elicit
an equivalent but minimal Ca2+
response that occurs immediately after serum addition. This could be
due to a variety of agonists found in serum. Nevertheless, this
stimulation does not inhibit further stimulation by
VEGF165 after either acute
exposure (Fig. 4, A and
B) or preincubation unless specific
antibodies against KDR are present (Fig. 3). Figure 4 also demonstrates
that a 30-min preincubation with the anti-KDR serum is required to
specifically attenuate the
VEGF165-mediated Ca2+ mobilization.

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Fig. 4.
Anti-KDR serum does not acutely stimulate KDR. HUVEC (~1 × 106) were loaded with fura 2-AM,
and
[Ca2+]i
was monitored. At 90 s (arrow at
left) either 1:100 dilution of
normal mouse serum (A) or 1:100
dilution of anti-KDR serum (B) was
added. Addition of VEGF165 (10 ng/ml) was made at 300 s (arrow at
right). Approximate
[Ca2+]i
was calculated according to Ref. 16.
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|
Our results from Fig. 3 suggested that Flt-1 was unable to mediate
Ca2+ mobilization in HUVEC. To
confirm this, we stimulated the endothelial cells with PlGF, a specific
ligand for Flt-1. Figure 5 represents one
of three experiments and shows that addition of up to 190 ng/ml PlGF
was without effect. Additionally, PCR analysis confirmed the
coexpression of both receptors in our cell cultures (data not shown).
Thus it appears that binding of PlGF to Flt-1 is not sufficient to
trigger an effective response. It has previously been reported that
PlGF can potentiate mitogenesis and permeability changes elicited by
submaximal VEGF165 stimulation
(19). In this assay, we determined that 0.4 ng/ml
VEGF165 was sufficient to elicit a
perceptible Ca2+ mobilization.
However, PlGF was unable to potentiate the response to this suboptimal
VEGF165 concentration.

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Fig. 5.
Effects of placental growth factor (PlGF) on
Ca2+ signaling in HUVEC. HUVEC
(~1 × 106) were loaded
with fura 2-AM, and
[Ca2+]i
was monitored. PlGF (190 ng/ml) and
VEGF165 (0.4 or 10 ng/ml) were
added at arrow. Approximate
[Ca2+]i
was calculated according to Ref. 16.
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|
VEGF can dimerize with itself to form homodimers or with PlGF to form
heterodimers. Although PlGF homodimers are specific for Flt-1, the
heterodimer is also capable of interacting with KDR. To our knowledge,
the effect of VEGF-PlGF heterodimer on Ca2+ signaling has not been
previously documented. We find that the heterodimer is unable to elicit
a Ca2+ response at 10 ng/ml, a
dose that is maximal for the
VEGF165 homodimer. Because the
heterodimer binds to KDR with a lower affinity than Flt-1, we raised
the concentration to 10- and 50-fold. Figure 6 shows that 100 ng/ml was ineffective and
that only a barely perceptible response was achieved at 500 ng/ml.

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Fig. 6.
Effects of VEGF-PlGF heterodimer on
Ca2+ signaling in HUVEC. HUVEC
(~1 × 106) were loaded
with fura 2-AM, and
[Ca2+]i
was monitored. VEGF (10 ng/ml) or VEGF-PlGF (100 or 500 ng/ml) was added at arrow. Approximate
[Ca2+]i
was calculated according to Ref. 16.
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|
 |
DISCUSSION |
In this study, we show that specific antiserum raised against the first
three immunoglobulin-like domains of KDR can inhibit VEGF165 binding to this receptor
and consequent signaling though Ca2+ in primary cultures of
endothelial cells. Our data confirm the location of the
VEGF165 binding determinants on
KDR that was recently reported by Kaplan et al. (15). In addition, they
are also consistent with the results of Davis-Smyth et al. (12), who
demonstrated that ligand binding activity was maintained on Flt-1 by
replacement of the second immunoglobulin-like domain with that of KDR.
Although both receptors interact with VEGF at high affinity, of the 21 amino acids on Flt-1 that make contact with VEGF in the crystal structure, only 1 is identical in KDR (32). It could be predicted that
a neutralizing antiserum raised against KDR would not inhibit VEGF
binding to Flt-1. Our data support this prediction.
With reports of additional uncharacterized VEGF binding proteins on
endothelial cells, monocytes, and pericytes, it is difficult to assign
specific signaling pathways to each receptor (27, 28). To address this,
several laboratories have constructed stable cell lines expressing
either Flt-1 alone or KDR alone. Although this approach has proven very
informative, it has drawbacks. One of the prerequisites for the choice
of cell line is an absence of basal expression of these receptors. This
also means that they may be missing other key endothelial cell-specific
signaling factors that engage with Flt-1/KDR upon receptor activation.
Indeed, this is the conclusion of Takahashi and Shibuya (29), who
compared transduction of signals from KDR to mitogen-activated protein kinase in endothelial cells vs. NIH/3T3 fibroblast stable cell lines.
We chose to address this concern by preparing specific neutralizing
antiserum against KDR and asking what effects the specific blockade of
this receptor has on VEGF165
signaling in primary cultures of endothelial cells. In this setting,
our data show that the binding of
VEGF165 to Flt-1 should remain
unperturbed. Furthermore, it is unlikely that this antibody would
inhibit the recently described interactions of
VEGF165 with the unrelated neuropilin-1 receptor (27).
It is clear that VEGF causes the tyrosine phosphorylation of PLC-
and mobilizes intracellular Ca2+
in HUVEC. Furthermore, VEGF induces membrane translocation of protein
kinase C (PKC) isoforms
,
II, and
in vivo (2). It is
not clear whether these signals are propagated simultaneously through
Flt-1 and KDR or whether signaling through one receptor predominates in
this pathway. In the yeast-two hybrid system, both Flt-1 and KDR
autophosphorylate tyrosine residues in the juxta-membrane and
carboxy-terminal tail that are capable of binding PLC-
(16).
Furthermore, stable cell lines expressing Flt-1 show that this receptor
can indeed utilize PLC-
as a signaling substrate (23). In contrast,
heterologous expression of KDR in PAEC lines does not reveal a role for
KDR in Ca2+ signaling (31).
Nevertheless, KDR is capable of mobilizing Ca2+ in
Xenopus oocytes (20) and can be
coprecipitated with PLC-
when expressed in NIH/3T3 cells (29). Most
importantly, KDR is capable of associating with PLC-
following its
activation in primary cultures of HUVEC (24).
In this study, we show that the
Ca2+ response elicited by
VEGF165 in primary cultures of
endothelial cells derived from the umbilical vein is primarily relayed
through the KDR receptor. It has previously been shown that
approximately one-half of the VEGF165 binding sites on HUVEC can
be competed against with PlGF (19) This suggests that Flt-1 is
expressed to levels at least as high as KDR in these cells (19). To our
knowledge, the only other study utilizing neutralizing antiserum to KDR
and primary cultures of endothelial cells focused on tissue factor
production (8). In combination with PlGF, these authors concluded that both receptors mediated this physiological response.
It has been reported that the full-length Flt-1 receptor is
inefficiently autophosphorylated both when expressed in its native endothelial setting and when recombinantly expressed in other cell
lines (19, 24). This is not a property of the intracellular domain per
se (10). Therefore, it appears that, despite very high affinity binding
to VEGF, Flt-1 is maintained in a repressed state. An inability to
efficiently dimerize or inhibition of autophosphorylation by
intracellular proteins must account for this phenomenon. An analogy
would be inhibition of the catalytic activity of the epidermal growth
factor receptor by PKC-mediated serine/threonine phosphorylation (7) or
association with caveolin (9). Alternatively, Flt-1 may recruit
phosphotyrosine phosphatases more efficiently. This inherent phenomenon
associated with Flt-1 may explain our results. However, because the
kinase domain possesses good potential binding sites for PLC-
, we
predict that, under defined conditions, Flt-1 has the capacity to
contribute to the Ca2+ response.
This is apparent in monocytes, in which Flt-1 can phosphorylate PLC-
and mobilize Ca2+ quite
efficiently (26). Thus the background expression of the cell is most
important. This is further demonstrated by the ability of PlGF to
elicit chemotactic and mitogenic effects to a greater degree in
coronary venular endothelial cells than in HUVEC (33).
Most interestingly, it is apparent that Flt-1 signaling is dependent on
the ligand with which it is activated. Landgren et al. (17) show that
Flt-1-expressing PAEC respond to PlGF with increased DNA synthesis, a
response lacking after VEGF stimulation. A weak growth stimulatory
effect of PlGF has also been documented in HUVEC (22). Nevertheless,
PlGF is unable to mediate a rapid transient intracellular
Ca2+ mobilization in our studies.
Potentiation of VEGF action by PlGF has previously been shown for
endothelial cell mitogenesis and permeability (19). This phenomenon
does not occur for Ca2+
mobilization in HUVEC.
We find furthermore, and to our surprise, that the VEGF-PlGF
heterodimer is extremely inefficient in signaling through
Ca2+ in HUVEC. This ligand can
bind both Flt-1 and KDR and induces the autophosphorylation of KDR in
HUVEC (5). However, it displays a much lower affinity for KDR, and this
translates to a 20- to 50-fold lower potency at inducing HUVEC cell
proliferation (5). Nevertheless, even at concentrations of 500 ng/ml, a
barely detectable Ca2+ response
could be measured. Thus, unless the heterodimer locally achieves these
high levels in vivo, it is unlikely to be a physiological mediator of
Ca2+ signaling. This suggests
that, like Flt-1, KDR may transmit some signals more efficiently than
others according to which ligand it interacts with. Furthermore,
because Flt-1 binds with high affinity to the VEGF-PlGF heterodimer, we
can conclude that binding does not translate to Flt-1 signaling through
Ca2+ in HUVEC. Finally, it should
be noted that, although we can conclude that KDR autophosphorylation is
crucial, we cannot rule out the possibility that the simultaneous
activation or heterodimerization of Flt-1 and KDR is required for
efficient signaling. To test this theory in the background of primary
endothelial cells, either specific neutralizing antiserum to Flt-1 or
specific activation of KDR alone is required.
 |
ACKNOWLEDGEMENTS |
We thank Kay Sughrue for maintenance and provision of HUVEC. We
also thank Dr. Richard Dixon for critically reading the manuscript.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: S. A. Cunningham, Dept. of Pharmacology,
Texas Biotechnology Corp., 7000 Fannin, Suite 1920, Houston, TX 77030.
Received 30 June 1998; accepted in final form 14 September 1998.
 |
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