© 2000 American Heart Association, Inc.
Vascular Biology |
Nitric Oxide Induces the Synthesis of Vascular Endothelial Growth Factor by Rat Vascular Smooth Muscle Cells
From the Department of Clinical Biochemistry (J.D., A.D-K., A.J., I.G., A.Z., D.Z-G., I.F., A.W.), Collegium Medicum, Jagiellonian University, Kraków, Poland; and the Falk Cardiovascular Research Center (A.S., J.P.C.), Stanford University, Stanford, Calif.
Correspondence to Dr Józef Dulak, Department of Cardiology, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail josef.dulak{at}uklibk.ac.at
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Abstract |
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Abstract—Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1ß (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N
-nitro-L-arginine methyl
ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP,
2.5 to 5 mmol/L), inhibitors of NOS and GTP
cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP
were supplemented with L-arginine (10 mmol/L) or
tetrahydrobiopterin (BH4; 100 µmol/L), respectively.
In addition, we studied the effect of sodium nitroprusside (SNP; 10 and
100 µmol/L) and chemically related compounds, potassium
ferrocyanide and ferricyanide, on VEGF generation. IL-1ß induced iNOS
expression and NO generation and significantly upregulated VEGF mRNA
expression and protein synthesis. L-NAME and DAHP totally inhibited NO
generation and decreased the IL-1ß–upregulated VEGF synthesis by
30% to 40%. Supplementation with L-arginine or
BH4 increased NO generation by L-NAME– or DAHP-treated
cells, and VEGF synthesis was augmented by addition of BH4.
The cells generating NO after pKecNOS transfection released
significantly higher amounts of VEGF than cells transfected with
control plasmids. Inhibition of NO generation by L-NAME decreased VEGF
synthesis. In contrast to the effect of endogenous NO, we
observed the inhibition of VEGF synthesis in the presence of high (10
or 100 µmol/L) concentrations of SNP. This effect was mimicked
by chemically related ferricyanide and ferrocyanide compounds,
suggesting that the inhibitory effect of sodium
nitroprusside may be mediated by an NO-independent mechanism. The
results indicate that endogenous NO enhances VEGF
synthesis. The positive interaction between endogenous NO
and VEGF may have implications for endothelial
regeneration after balloon angioplasty and for angiogenesis.
Key Words: VEGF • nitric oxide • atherosclerosis • tetrahydrobiopterin • gene transfer
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Introduction |
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Vascular endothelial growth factor (VEGF; VEGF-A) induces proliferation of endothelial cells and increases permeability of the vessel wall. VEGF-A belongs to a group of related growth factors that also includes VEGF-B, VEGF-C, and placenta-derived growth factor (PlGF).1 The physiological activity of VEGF-A is exerted via the specific tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk1/KDR) present particularly on endothelial cells.2 3
VEGF-A is produced by different cell types, including vascular smooth muscle cells (VSMCs), macrophages, fibroblasts, tumor cells, and endothelial cells.1 4 5 6 7 8 9 10 The expression of several known VEGF-A isoforms is induced by hypoxia; growth factors, including transforming growth factor-ß, basic fibroblast growth factor, and platelet-derived growth factor; and cytokines, among them interleukin (IL)-1ß and IL-6.6 7 8 9 10 It has recently been demonstrated that the physiological effects of VEGF-A are mediated in part by endothelium-derived nitric oxide (NO).11 12 13 Previous studies have shown that enhancement of vascular NO activity after balloon angioplasty suppresses neointimal hyperplasia, an effect that has been ascribed to the inhibitory influence of NO on VSMC growth.14 15 However, it is also possible that NO (through VEGF) might enhance endothelial regeneration, thereby reducing restenosis.
The phosphorylation of the flk-1 receptor induces NO
release, followed by activation of ERK-1/2 kinase in
endothelial cells.13 The VEGF-dependent
release of NO and angiogenic activity of VEGF-A is blocked by tyrosine
kinase inhibitors16 17 as well as by
N-nitro-L-arginine
methyl ester (L-NAME),16 17 an NO synthase (NOS)
inhibitor. The vital role of NO has been demonstrated in
eNOS knockout mice, in which angiogenesis is impaired.18
Furthermore, the circulating inhibitor of NOS asymmetric
dimethylarginine (ADMA) impairs angiogenesis19 ; its
elevation in hypercholesterolemia and
atherosclerosis may explain the impairment of
angiogenesis in these diseases.20
Recently it was demonstrated that NO donors inhibit VEGF expression in rat VSMCs, although inhibition occurs at pharmacological concentrations of NO donors.21 22 In contrast, exogenous NO donors had a stimulatory effect on VEGF synthesis by certain tumor cell lines.23 Therefore, the physiological effect of endogenous NO on VEGF expression has not been clarified.
Accordingly, we performed this study to determine the effects of endogenous and exogenous NO on VEGF synthesis.
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Methods |
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Reagents
DMEM F-12 medium and FCS were purchased from Gibco BRL, and the Total RNA Extraction Kit, Tth DNA polymerase, pSVßgal plasmid, and TFx-50 liposomes from Promega. Primers were synthesized by TIBMOLBIOL or ARK. Lactate dehydrogenase (LDH) assay was obtained from Wako; reagents for nonradioactive hybridization were obtained from Boehringer Mannheim; and all other chemicals were purchased from Sigma. VEGF concentration was measured by an ELISA kit for mouse VEGF (R&D) that also recognizes rat VEGF.
Plasmid Isolation
The pKecNOS expression plasmid, with kanamycin resistance gene
and containing 4.2 kb of bovine endothelial cNOS cDNA,
driven by cytomegalovirus (CMV) promoter, was kindly provided by Dr
Thomas Michel (Boston, Mass). The plasmids were amplified in
Escherichia coli and isolated with Qiagen Endo-Free plasmid
kit, which provides supercoiled plasmid with <1 ng of LPS/mL plasmid
solution. The DNA purity was confirmed spectrophotometrically and by
agarose electrophoresis.
The following control plasmids were used: pSVßgal expression plasmid (contains bacterial ß-galactosidase gene under the control of the SV40 promoter), pCISGTPCH plasmid (contains GTP cyclohydrolase I cDNA driven by the CMV promoter, kindly provided by Drs Edith Tzeng and Timothy Billiar, Pittsburgh, Pa), and piNOSCAT plasmid (containing the CAT gene under the control of the rat inducible NOS [iNOS] promoter, kindly provided by Dr Josef Pfeilschifter, Frankfurt am Main, Germany).
Cell Culture
VSMCs were isolated by collagenase digestion
of rat thoracic artery and cultured in DMEM F-12 with 5% FCS at 37°C
in an atmosphere of 5% CO2. Cells of the 5th to
10th passages were used for experiments.
Experimental Procedure
Induction of iNOS Expression
The cells were cultured to full confluence and then placed in
DMEM F-12 medium with 0.5% FCS for 24 hours (the presence of a low
concentration of FCS is necessary to stabilize VEGF released into the
medium). Subsequently, the cells were exposed to IL-1ß (10 ng/mL) for
24 hours. In some experiments, the cells were exposed to IL-1ß in the
presence of the NOS inhibitor L-NAME (2 to 5 mmol/L)
or the GTP cyclohydrolase I inhibitor
diaminohydroxypyrimidine (DAHP; 2.5 to 5 mmol/L). Some cells
exposed to L-NAME or DAHP for 24 hours were also supplemented with
L-arginine (10 mmol/L) or tetrahydrobiopterin
(BH4; 100 µmol/L), respectively.
Alternatively, cells were treated with sodium nitroprusside (SNP), K4Fe(CN)6, or K3Fe(CN)6 (10 or 100 µmol/L) for 48 hours.
Transfection of VSMCs
For the purpose of gene transfection, cells were cultured in
DMEM F-12 with 5% FCS to 50% to 70% confluence. Two liposomes were
used for these experiments. The Maxifectin liposome (kindly provided by
Dr Andrey Surovoy, Rottenburg, Germany) was used for transfection in
the presence of 5% serum, and the Tfx-50 lipotransfection was
performed in the absence of serum. Initially, experiments were
performed with Maxifectin liposome, and later, all experiments were
repeated with Tfx-50 reagent.
Maxifectin lipotransfection: The VSMCs at 50% to 70% confluence were transfected with 1 µg of plasmid DNA/30-mm well. The DNA was mixed with 2 µL of Enhancer and 10 µL of Maxifectin in 200 µL of binding buffer (10 mmol/L HEPES, 0.9% NaCl, pH 7.4). After a 20-minute incubation period, the transfection mixture was added to cells covered with 1.8 mL of fresh DMEM F-12 with 5% FCS. The cells were exposed to the transfection mixture for 24 to 48 hours.
Tfx-50 lipotransfection: Plasmid DNA (2.5 µg) was mixed with 1 mL of DMEM F-12 without serum, and Tfx-50 was added in a proportion of 2:1 according to the manufacturer’s protocol. After 15 minutes of incubation at room temperature, the transfection mixture was poured onto the cells. After 1 hour of incubation at 37°C, 1 mL of DMEM F-12 with 10% FCS was added to the cells, and the VSMCs were incubated up to 24 or 48 hours.
The following experimental groups were established: control cells, which were not transfected; cells treated with liposomes only; cells stimulated with LPS (100 ng/mL) in the presence or absence of liposomes; and cells transfected with pKecNOS, pSVßal, plSGTPCH, or piNOSCAT plasmid. The pKecNOS-transfected cells were additionally treated with BH4 (100 µmol/L) and/or L-NAME (2 mmol/L).
Measurement of VEGF and NO Synthesis
VEGF synthesis by control, transfected, or IL-1ß–treated
cells was measured by ELISA in the medium collected 24 or 48 hours
after stimulation.
The generation of NO was detected by Griess reaction24 for the cells treated with IL-1ß. The more sensitive fluorometric method (detects nitrite at 0.07 to 10 µmol/L)25 was used for estimation of NO2- generation by transfected VSMCs.
Expression of VEGF and iNOS mRNA
Isolation of RNA
Total cellular RNA was isolated from VSMCs according to the
method of Chomczynski and Sacchi26 with a Total RNA
Extraction Kit. The RNA concentration and purity were assessed
spectrophotometrically by the optical density measured at 260/280 nm.
RNA was diluted in RNase-free water and kept at -70°C.
Primers
The iNOS-specific primers (see the
Table
) were used to generate a
384-bp product. The primers for VEGF (Table) generated
different products, depending on the kind of VEGF isoform
expressed, namely a 431-bp product for
VEGF120 (the rat equivalent of human
VEGF121), 563 bp for
VEGF164, and 635 bp for
VEGF188. Primers for the rat GAPDH (Table)
(housekeeping, reference gene) were used to produce a 452-bp
product as a control for RNA isolation and amplification.
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Reverse Transcription–Polymerase Chain Reaction
A qualitative analysis of mRNA expression was performed
by means of reverse transcription–polymerase chain reaction (RT-PCR)
assay using 500 ng of total RNA obtained from cells at different time
points after the addition of IL-1ß. A 20-minute RT step was performed
at 70°C (1 U Tth DNA polymerase, 1 mmol/L
MnCl2, 1 µmol/L downstream [3'] primer).
After that, 20 µL of chelating buffer (750 µmol/L EGTA, 0.5 U
PrimeZyme, 2.5 mmol/L MgCl2, 250 nmol/L
upstream [5'] primer) was added, and 30 cycles of PCR were performed
(94°C for 20 seconds, 62.5°C for 20 seconds, 72°C for 20 seconds)
in UNO Thermoblock (Biometra). PCR products were analyzed
electrophoretically on ethidium bromide–stained agarose gel.
Nonradioactive Dot-Blot Hybridization
Hybridization with antisense, digoxigenin-labeled VEGF
DNA probe generated during 1-primer PCR was performed in a Micro-4
Hybridization oven (Hybaid). Nylon membranes with bound RNA (10 µg of
each sample) were prehybridized for 2 hours at 42°C in a high-SDS
hybridization buffer (7% SDS, 5xSSC, 1% blocking reagent, 50%
formamide, pH 7.0). After that, the digoxigenin-labeled probes were
added to a hybridization buffer. The concentrations of probes were
established in 20 to 30 ng/mL buffer. Hybridization was performed
overnight at 42°C. The next day, the membranes were washed twice with
2xSSC, 0.1% SDS at room temperature, followed by a 2-fold wash with
0.1xSSC, 0.1% SDS at hybridization temperature and blocked with
excess amounts of 1% blocking reagent. Immunodetection was performed
at room temperature for 1.5 hours with alkaline phosphatase–labeled
anti-digoxigenin antibody (1:3000 to 1:5000 stock dilution in 1%
blocking reagent). The hybridization was revealed by overnight reaction
with the alkaline phosphatase substrate NBT/X-phosphate diluted in
buffer (Tris-HCl 100 mmol/L, NaCl 100 mmol/L,
MgCl2 50 mmol/L, pH 9.5).
Protein Estimation
The cells were washed twice with PBS, scraped off, and lysed
with NaOH (1 mol/L), and total protein was estimated by the Lowry
method.27
Estimation of Cell Viability
The cell viability was assessed by LDH release assay according
to the vendor’s protocol.
Statistical Methods
Data are presented as mean±SD. Statistical evaluation
was done with ANOVA followed by Tukey’s test. A value of
P<0.05 was accepted as statistically significant.
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Results |
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VEGF Synthesis in IL-1ß–Stimulated VSMCs Is Decreased by NOS Inhibitors
The expression of iNOS and VEGF mRNA was induced by IL-1ß (Figure 1
). In control cells, which were
not exposed to IL-1ß, only weak expression of
VEGF164 and VEGF120
isoforms was observed (Figure 1); IL-1ß upregulated the
expression of both isoforms. Twenty-four hours after IL-1ß
stimulation, the expression of the third isoform,
VEGF188, was also observed (Figure 1).
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Treatment with IL-1ß induced VSMCs to generate NO (Figure 2A) and to secrete significantly more
VEGF protein (up to 400 pg/mL) into the medium than control VSMCs (up
to 140 pg/mL) (Figure 2B), depending on the cell batch (Figure 2B). L-NAME inhibited NO production (Figure 2A)
and reduced IL-1ß–induced VEGF synthesis 
30% to 40% (Figure 2B). Similarly, inhibition of NO generation by DAHP (Figure 3A) also reduced the synthesis of VEGF by
VSMCs (Figure 3B).
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Supplementation with L-arginine did not significantly
enhance NO generation by L-NAME–treated cells (Figure 2A
) or
the synthesis of VEGF (Figure 2B). Increased VEGF synthesis was
observed in cells treated with IL-1ß and L-arginine
(Figure 2B). Supplementation of DAHP-treated cells with
BH4 slightly increased NO generation (Figure 3A) but strongly upregulated VEGF synthesis (Figure 3B).
Control cells, when exposed to BH4, did not
increase VEGF synthesis (Figure 3B). Neither L-NAME (Figure 2B) nor DAHP (Figure 3B) alone significantly influenced
basal VEGF synthesis.
Generation of NO by pKecNOS-Transfected VSMCs Results in VEGF
Expression and Synthesis
When VSMCs were transfected with pKecNOS, they generated NO
and expressed VEGF mRNA, as demonstrated by dot-blot hybridization
(Figure 4A) and RT-PCR (Figure 4B). In control cells or in cells transfected with control
plasmids, only weak VEGF expression was observed (Figure 4, A
and B).
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The amount of NO generated by pKecNOS-transfected VSMCs ranged from 10
to 100 nmol NO2-/mg cellular
protein (Figure 5A); the
production of NO was inhibited by L-NAME (Figure 5A).
Significantly lower NO2-
concentrations were observed in cells transfected with pSVßgal
(Figure 5A), pClSGTPCH I, or piNOSCAT control plasmids as well
as in VSMCs treated with 100 ng/mL LPS (data not shown).
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Cells transfected with pKecNOS plasmid generated significantly higher
amounts of VEGF protein than cells transfected with control plasmids
(pSVßgal, pISGTPCH, or piNOSCAT) or treated with liposomes (Figure 5B) or LPS (not shown). L-NAME significantly inhibited NO
generation by pKecNOS-transfected VSMCs (Figure 5A) and reduced
VEGF synthesis by these cells (Figure 5B).
The transfection procedure did not affect cell viability, as demonstrated by LDH measurements of the conditioned medium (data not shown).
Effect of NO Donors and Ferrocyanides
Incubation of VSMCs with SNP led to accumulation of nitrites in
culture medium in concentrations similar to those obtained after
IL-1ß stimulation (up to 30 µmol/L). This effect was not
observed for 2 structurally similar compounds, potassium ferrocyanide
and ferricyanide, that are not NO donors. After 48 hours of incubation
with SNP (100 µmol/L), VEGF generation by VSMCs was
significantly decreased. This inhibitory effect was
partially mimicked by ferrocyanide and ferricyanide and was correlated
with LDH levels in the conditioned medium (r=0.92,
P<0.003) (Figure 6).
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Discussion |
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Previous studies have shown that VEGF expression is induced in VSMCs by IL-1ß.28 29 This cytokine triggers the expression of iNOS and generation of NO by VSMCs.29 30 31 32 Earlier results from our laboratory revealed that iNOS induction after balloon angioplasty of the rat aorta is followed by VEGF expression.31 In the present study, we determined that VEGF induction by IL-1ß was related to iNOS activity in VSMCs. We also determined that expression of transfected endothelial constitutive NOS (ecNOS) gene and its resulting production of NO induces VEGF expression in rat VSMCs. Furthermore, when NO generation by NOS was inhibited by L-NAME or DAHP, VEGF synthesis was concomitantly decreased. Finally, when NO generation was even slightly increased by L-arginine or BH4 supplementation, VEGF synthesis was also restored. Thus, NO generated by VSMCs appears to exert an autocrine or paracrine effect on VSMCs to enhance VEGF synthesis.
Our results are consistent with previous observations that IL-1ß may induce the generation of NO30 32 or VEGF28 29 by smooth muscle cells. However, we show for the first time that NO synthesis augments VEGF expression in these cells. The mechanism by which NO enhances VEGF synthesis is under investigation; our results suggest that NO enhances the transcription or stability of VEGF mRNA.
In our hands, L-NAME completely inhibited NO synthesis and diminished VEGF generation in IL-ß–stimulated VSMCs. However, despite cessation of NO synthesis, the VEGF generation was still higher than in control, untreated cells. This indicates the existence of an additional, NO-independent regulation of VEGF synthesis.
Reactive oxygen intermediates are known to stimulate VEGF synthesis in retinal pigment epithelial cells and in some cancer cells.33 However, it is not likely that superoxide radicals generated by NOS were responsible for the observed effects. We observed that VEGF was further upregulated in the presence of BH4 and L-arginine, which are both known to reduce the superoxide generation by NOS.34 35 36 However, BH4 seems to increase VEGF synthesis partially independently of NO generation. We observed that BH4 restores or even upregulates VEGF synthesis but not NO generation in VSMCs treated with IL-1ß or IL-1ß and DAHP. A similar effect of BH4 on VEGF synthesis was also observed in VSMCs transfected with ecNOS (data not shown). Thus, the role of BH4 in regulation of this growth factor synthesis requires further investigation.
Recent studies performed by Tsurumi and coworkers21 suggested an interaction between VEGF and NO generation in the vessel wall. According to their work, VEGF released by the VSMCs of a damaged artery may stimulate reendothelialization, acting together with NO. This suggestion is consistent with studies showing that VEGF protein37 or VEGF cDNA transfection38 inhibits restenosis. However, Tsurumi et al found that VEGF expression was downregulated by a high concentration of NO donors.21 They suggested that inhibition of VEGF synthesis may occur when endothelial regeneration is finished.21
We have also observed an inhibition of VEGF generation by pharmacological doses of NO donors (Reference 2222 , this study, and a study in preparation). However, the inhibitory effect of SNP can be partially mimicked by the same amounts of chemically related compounds, potassium ferrocyanide and ferricyanide, that are not NO donors. The pharmacological doses of these compounds were cytotoxic for rat VSMCs, as indicated by LDH release, and LDH levels were correlated with the decrease in VEGF generation. Thus, although NO concentration was indeed high in medium of cells treated with SNP, it seems likely that the inhibition of VEGF synthesis was related to the toxicity of cyanides released by SNP and the ferrocyanide and ferricyanide.39
Other recent studies support our conclusion that NO can upregulate VEGF
synthesis. Xiong and coworkers40 demonstrated in murine
RAW264.7 macrophages that NOS inhibitors blocked
interferon-
/LPS–activated VEGF
production.40 In studies performed on tumor cell
lines, Chin et al23 observed that NO donors increased the
stability of VEGF mRNA. Increased VEGF synthesis by cancer cells
generating NO has also been demonstrated by Ambs and
coworkers,41 42 and upregulation of both NO and
BH4 synthesis in skin wounds was correlated to
increased VEGF production.43 These results are in
accordance with our recent observations demonstrating that
endogenously generated NO can upregulate VEGF synthesis in
VSMCs. Finally, recent studies reported by Gallacher et
al44 demonstrated that in human VSMCs, the NO donors
S-nitro-N-acetyl-penicillamine and SNP increased
VEGF expression and synthesis at high concentration, whereas low doses
of those NO donors inhibited VEGF generation.44 The
reason for the partial discrepancy between the results of the latter
and our study is not yet known. One may suggest that the effect of NO
donors is slightly different in human than rat VSMCs. However, the
NO-dependent upregulation of VEGF synthesis seems to be a general
phenomenon, because our recent data (unpublished) indicate that
endogenous NO generated by ecNOS-transfected human
coronary artery VSMCs induced VEGF synthesis in the same way as
it did in rat VSMCs. It is also possible that the effects of NO donors
can depend on the cell culture conditions.
In the present investigations, we have demonstrated that transfection with pKecNOS plasmid resulted not only in the generation of NO by VSMCs but also in VEGF synthesis in such cells. The existence of such a relationship adds to the understanding of the role of ecNOS-derived NO in the regulation of proliferation of endothelial cells. Although some studies demonstrated that NO may stimulate the proliferation of endothelial cells,11 12 13 45 others failed to prove such a mechanism or even suggested an inhibitory effect of NO46 on endothelial proliferation. It is known that transfer of eNOS14 or iNOS15 genes resulted in the inhibition of restenosis after balloon angioplasty. The inhibitory activity of NO on VSMC proliferation was suggested to exert this protective effect.14 15 However, our recent results also indicate that the upregulation of VEGF synthesis by ecNOS-transfected VSMCs might improve reendothelialization in the denuded arteries.14 15 Similarly, L-arginine supplementation after balloon angioplasty of rabbit iliac arteries was demonstrated to be beneficial for healing of endothelium,47 which according to our results may be related to the increased VEGF synthesis due to the enhanced NO generation.
The observed increase in VEGF synthesis after ecNOS transfection cannot be ascribed to contamination of the plasmid isolation by LPS. In our hands, even high doses of LPS did not induce either iNOS or VEGF expression in rat VSMCs, in accordance with earlier observations.48 We have also shown that the stimulatory effect of transfection itself on VEGF synthesis is not related to the DNA backbone of the control plasmid used, because we observed similar results with different constructs. The mechanism is not clear, and we may only speculate that some degradation products of plasmid DNA can influence VEGF synthesis.
To conclude, we find that endogenous NO enhances VEGF expression by VSMCs. The induction of VEGF synthesis by NO may be of great importance in the maintenance of vascular homeostasis and in the response to endothelial injury. Because NO and VEGF reciprocally enhance their synthesis, this interaction may play a significant role in reendothelialization after balloon angioplasty or in the angiogenic response to ischemia.
Note Added in Proof
Recently Kimura et al49 demonstrated that some
NO-donors upregulated the activity of the human VEGF promoter in human
glioblastoma and hepatoma cells, independently of a
cGMP-mediated pathway.
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Acknowledgments |
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This research was supported by grants from the Polish State Committee for Scientific Research (4 P05A 131 14 and 4 P05A 108 17) awarded to J. Dulak and A. Józkowicz, respectively.
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Footnotes |
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Drs Dulak and Józkowicz contributed equally to this work.
Received November 25, 1998; accepted October 11, 1999.
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