© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Overexpression of 15-Lipoxygenase in Vascular Endothelium Accelerates Early Atherosclerosis in LDL Receptor–Deficient Mice
From the Institute of Lipid & Atherosclerosis Research (D.H., A.S., J.G., H.L.), Sheba Medical Center, Tel-Hashomer, Israel; Bristol-Myers Squibb (M.M., E.S.), Princeton, NJ; and the Third Department of Medicine (H.K.), University of Tokyo, Hongo, Tokyo, Japan.
Correspondence to Dror Harats, MD, Institute of Lipid & Atherosclerosis Research, Sheba Medical Center, Tel-Hashomer, 52621 Israel. E-mail dharats{at}post.tau.ac.il
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Abstract—To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor–deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO–overexpressing, LDL receptor–deficient mice (LDLR-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor–deficient (LDLR-/-) mice after 3 and 6 weeks (107 000 versus 28 000 µm2 [P<0.001] and 121 000 versus 87 000 µm2 [P<0.05], respectively) of an atherogenic diet. LDL from the LDLR-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti–oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.
Key Words: 15-lipoxygenase • oxidation • endothelium • gene expression • atherosclerosis
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Introduction |
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Early atherosclerotic lesions are characterized by lipid-laden foam cells in the arterial intima.1 There is now ample evidence to suggest that LDL oxidation occurs in vivo in the vicinity of atherosclerotic plaque.2 3 Many mechanisms, enzymatic and nonenzymatic, have been proposed for the oxidation of LDL in the atherosclerotic plaque. Cellular lipoxygenases (LOs) have been suggested to be involved in LDL modification by endothelial cells and macrophages.4 5 15-LO has been suggested to be a mediator of oxidation in vivo,6 although other enzymes, such as myeloperoxidase, have been suggested as well.7
Human and rabbit 15-LOs, as well as the leukocyte-type 12-LO, are unique in their ability to oxidize fatty acids esterified to membranes and LDL.4 8 9 The 15-LO enzyme forms hydroperoxy derivatives of linoleic acid (13-hydroperoxyoctadecadienoic acid) and arachidonic acid (15-hydroperoxyeicosatetraenoic acid) and is induced in atherosclerotic plaques.10
Several lines of evidence suggest the involvement of 15-LO in LDL oxidation. It has been implicated in the oxidative modification of LDL in cultured endothelial cells and in monocytes.4 11 Soybean 15-LO incubated with LDL in the presence of phospholipase A2 oxidizes LDL, which is recognized by the scavenger receptor.12 Moreover, human 15-LO has been shown to oxidize LDL without requiring a phospholipase,13 and fibroblasts that overexpress 15-LO generate minimally modified LDL with bioactive properties.14 The enzyme expression and activity in rabbit and human lesions15 16 17 18 19 provide the most convincing evidence for the localization of the enzyme in the atherosclerotic lesion. Moreover, elevated activity was found throughout the aortas of Watanabe and cholesterol-fed rabbits, suggesting that the enzyme may be a response to hypercholesterolemia.20 Hence, 15-LO may be induced in the vessel wall early in atherogenesis before plaque is formed.
Because 15-LO is expressed in the vascular wall, it can promote atherogenesis by altering endothelial cell function. Several works indicate that 15-LO is proatherogenic. It has been implicated in endothelial cell oxidation of LDL,4 and the enzyme metabolites have been shown to cause injury to cultured endothelial cells,21 to induce the expression of adhesion molecules on human umbilical vein endothelial cells,22 and to bring about the appearance of LDL oxidation products in rabbit iliac arteries.23 Moreover, a recent study shows that disruption of the 12/15-LO gene diminishes atherosclerosis in apoE-deficient mice.24 Sparrow and Olszewski,25 who used the dual cyclooxygenase-LO inhibitor, found discrepancies between the drug concentrations required to inhibit the oxidation of free fatty acid and those required to inhibit LDL modification by murine macrophages. However, studies with 15-LO–specific inhibitors lacking significant antioxidant activity have suggested that the enzyme is involved in atherogenesis.26 27 In contrast to the proatherogenic effects of 15-LO, several studies report that the enzyme metabolite 13-hydroxyoctadecadienoic acid has antiatherogenic activities.28 29 30 The antiatherogenic activity of 15-LO was demonstrated in vivo by Shen and colleagues.31 32 They developed transgenic New Zealand White rabbits and heterozygous Watanabe heritable hyperlipidemic rabbits with integrated human 15-LO, driven by the lysozyme promoter. Surprisingly, although the 2 lines of transgenic rabbit showed 15-LO overexpression in macrophages, atherosclerotic lesion development was reduced in the transgenic animals.
To investigate further the relation of 15-LO to atherosclerosis in the mouse, we created vascular-specific human 15-LO–overexpressing transgenic mice by using the murine prepoendothelin-1 promoter.33 In the present study, we used murine prepoendothelin-1 promoter for overexpressing 15-LO specifically in the vascular wall of LDL receptor–deficient (LDLR-/-) mice and studied its effect on atherogenesis.
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Methods |
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LDLR-/-/15LO Double-Transgenic Mice
To establish a colony of LDLR-/- mice with human 15-LO activity derived with the preproendothelin promoter (LDLR-/-/15LO mice), we crossbred LDLR-/- mice34 with 2 independent homozygous colonies of preproendothelin-1 promoter–15-LO–overexpressing mice with a background of C57B6/SJL.33 Double-transgenic mouse colonies were established and screened by polymerase chain reaction with use of primers for both transgenes and the wild-type LDL receptor gene as described.6
Experimental Design
Two sets of experiments were performed. In each experiment,
forty-five 3-month-old mice in each group were studied. The mice were
fed a high-cholesterol high-fat diet containing 15.75% fat
(43% saturated fat), 1.25% cholesterol, and 0.5% sodium
cholate (Harlan, Teklad). To minimize oxidation of
cholesterol and lipids, the diet was kept in a cold room at
4°C and fed daily ad libitum. Animals from each group were euthanized
at 0, 3, and 6 weeks in the first experiment and at 0, 3, and 9 weeks
in the second experiment. Fifteen mice from each group were killed at
each time point. The Sheba Medical Center Animal Studies Committee
(Tel-Hashomer, Israel) approved all procedures.
Plasma Lipid Levels
Cholesterol and triglyceride levels were
measured with an enzymatic kit (Boehringer-Mannheim) at 0, 3,
6, and 9 weeks.
Lipoprotein Oxidation
LDLs (fraction 1.019 to 1.063) were isolated from pooled plasma
of 9 mice from each group, and 3 pools from each group were
analyzed separately. Lipoproteins were incubated at a
concentration of 50 µg/mL PBS, pH 7.4, with 15 µmol/L
CuSO4. Incubation was carried out at 37°C in
the dark. Lipid oxidation was measured as conjugated diene formation at
234-nm wavelength.35
Macrophage Preparation
Peritoneal macrophages were isolated and purified as
previously described.36 Briefly, macrophages in
LDLR-/- and
LDLR-/-/15LO mice were
elicited by intraperitoneal injection of 2.4%
Brewer thioglycollate medium (2 mL, Difco Laboratories). Three days
later, the peritoneal macrophages were harvested by 2 mL PBS
lavage. Washed cells, 1x106 in a total volume of
2 mL, were plated on 35-mm-diameter polystyrene tissue-culture Petri
dishes with DMEM (Biological Industries) supplemented with 20%
heat-inactivated FCS. The cells were incubated for 2 hours
at 37°C in 5% CO2/95% air. Adherent
macrophages were washed twice with DMEM, collected after
trypsin treatment, pelleted at 2000 rpm, centrifuged for 5
minutes, and resuspended in 1 mL of cold PBS containing 5 mmol/L
glucose, pH 7.4.
15-LO Enzymatic Activity
To assess the level of expression of the human 15-LO in the
double-transgenic mice, we performed an enzyme activity assay in the
mouse tissues and in isolated peritoneal macrophages. We
measured the enzyme product,
15-hydroxyeicosatetraenoic acid
(15-HETE), by a standard high-performance liquid
chromatography (HPLC) technique.33
Briefly, mice were euthanized, and their organs were harvested, trimmed
of fat and connective tissue, weighed, minced, and resuspended in 1 mL
of cold PBS containing 0.5 mmol/L glucose, pH 7.4. For each
activity assay, 200 mg of tissue was used. The reactions were carried
out in a total volume of 1 mL at 37°C for 15 minutes, with 20
µmol/L arachidonic acid used as a substrate. The
reaction was terminated with 100 µL glacial acetic acid, and the
lipids were extracted with 1 vol isopropyl alcohol and 1 vol
chloroform. An aliquot of prostaglandin
B2 was used as an internal standard. All extracts
were dried under N2 and stored at -70°C.
Extracts were reconstituted in chromatography solvent
and were analyzed by reverse-phase HPLC on a
chromatography system (Kontron Instruments Inc) with
use of an Adsorbosil C18 column (Vydac 201TP-54; 250x5 mm, 5-µm
particle size). The column was developed at a flow rate of 1.0 mL/min
by an isocratic solvent system,
methanol/H2O/glacial acetic acid (850:150:0.1
[vol/vol/vol]). The eluate was monitored with a Kontron 430 HPLC
detector.
Assessment of Atherosclerosis in the Aortic
Sinus
Quantification of atherosclerotic fatty-streak lesions was
performed by measuring the lesion size in the aortic sinus. The heart
and upper section of the aorta were removed from the animals, and the
peripheral fat was cleaned carefully. The upper section was
embedded in OCT compound (Miles Inc) and frozen. Every other section
(10 µm thick) throughout the aortic sinus (400 µm) was
taken for analysis. The distal portion of the aortic sinus was
recognized by the 3 valve cusps that constitute the junctions of the
aorta to the heart. Sections were evaluated for fatty-streak lesions
after they were stained with oil red O. Lesion areas per sections were
counted with use of a grid by an observer unfamiliar with the tested
specimen.
Sudan IV Staining of Aortic Lesions
The aortas were dissected from the aortic arch to the iliac
bifurcation and washed 1 for hour in PBS, pH 7.4, and 0.5 mmol/L
EDTA on a rotating table. The aorta was then fixed with formal-sucrose
(4% paraformaldehyde, 5% sucrose, 20 mmol/L
butylated hydroxytoluene, and 2 mmol/L EDTA, pH 7.4) overnight.
The adventitial fat was trimmed from the aorta under a microscope and
opened longitudinally, rinsed briefly in 70% ethanol, immersed for 6
minutes in a filtered solution of Sudan IV (Sigma Chemical Co) in 35%
ethanol and 50% acetone for 10 minutes, and destained in 80%
ethanol.37 The Sudan IV–stained aortas were placed on a
microscope slide and photographed. Lesion area was detected by
morphometry.
Detection of Anti-OxLDL Antibodies by ELISA
Polystyrene plates with 96 wells (Nunc Maxisorp) were coated
with either copper-induced oxidized LDL (oxLDL, at a concentration of
10 µg/mL in PBS) or native LDL overnight at 4°C. The plates were
washed 4 times with PBS containing 0.05% Tween and 0.001% aprotinin
(Sigma) and then blocked with 2% BSA for 2 hours at room temperature.
Diluted (1:50) serum fractions were added in PBS containing 0.05%
Tween and 0.2% BSA. The plates were incubated at 4°C overnight, the
sera were washed, and alkaline phosphatase–conjugated goat anti-mouse
IgG (Jackson Immuno-Research Laboratory Inc) was added (diluted
1:10 000 in PBS, 0.05% Tween, and 0.2% BSA) for 1 hour at room
temperature. The plates were washed again, and 1 mg/mL
p-nitrophenylphosphate (Sigma) in 50 mmol/L carbonate
buffer containing 1 mmol/L MgCl2, pH 9.8,
was added as a substrate. The reaction was stopped at 30 minutes by
adding 1 mol/L of NaOH. Absorbance was detected at a 405-nm wavelength
in a Titertek ELISA reader (S.L.T Laboratory Instruments), and results
are expressed as absorbance at 405 nm. Anti-oxLDL levels were
calculated as binding to native LDL subtracted from oxLDL binding.
Immunohistochemistry
Immunohistochemical staining was performed by use of anti-CD4
(rat anti-mouse, clone H129.19 [L3T4]) and CD8a (clone S3-6.7
[Ly-2]) from PharMingen and macrophages (rat anti-mouse MCA
497 [F4/80]) from Serotec. Anti–malondialdehyde-LDL antibodies were
obtained by immunization of mice with homologous malondialdehyde-LDL
and performed by use of the Histomouse-SP-Bulk-Kit (Zymed-Laboratory
Inc) for detection of mouse primaries on mouse tissues. The
immunohistochemical studies were performed on 5-µm-thick frozen
sections of the aortic sinus. The sections were fixed for 4 minutes in
methanol at -20°C, followed by 10 minutes of incubation with ethanol
at -20°C. The sections were then blocked with nonimmune goat serum
for 15 minutes at room temperature, followed by incubation with CAS
blocking reagent (Zymed) for 30 minutes at room temperature subsequent
to incubation with biotinylated antibodies. After they were washed, the
slides were incubated in 0.3%
H2O2, followed by
additional rinses, and developed with peroxidase streptavidin complex.
Sections were counterstained with hematoxylin. Spleen sections were
used as a positive control. Staining in the absence of first or second
antibody was used as a negative control.
To determine the tissue distribution and cellular localization of 15-LO expression, primary organs and the aortic sinus were prepared for paraffin-embedded sections. Immunoperoxidase staining was assessed on 5-µm sections prepared from formalin-fixed paraffin-embedded tissues. Sections were deparaffinized and permeabilized with PBS containing 0.2% Nonidet P-40 detergent (Sigma). Sections were then immersed sequentially in PBS containing 0.5% BSA and 10% normal goat sera (blocking solution) for 10 minutes, followed by blocking solution containing 10 µg/mL avidin for 10 minutes, blocking solution containing 10 µg/mL biotin for 10 minutes, and primary polyclonal rabbit anti-human 15-LO antibody, raised against the native enzyme, at 1:2000 blocking solution (overnight at 4°C). The sections were then incubated with biotinylated goat anti-rabbit IgG (1:250, Vector Labs), and endogenous peroxidase activity was then quenched with 3% H2O2 for 5 minutes. Bound primary antibody was detected by ABC (Vector Labs), followed by the substrate, aminoethyl-carbazole (Vector Labs) or diaminobenzidine (Vector Labs), and counterstained with hematoxylin.
Statistical Analyses
All values are reported as mean±SE. Statistical
analyses were performed by Student t test.
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Results |
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15-LO Expression in the Double-Transgenic Mice
To determine the effect of 15-LO activity in the vascular wall on atherogenesis, we have produced transgenic LDLR-/- mice overexpressing human 15-LO activity under the control of the endothelial cell–specific promoter, preproendothelin (LDLR-/-/15LO mice). The double-transgenic mice were morphologically normal and healthy. 15-LO expression and activity in the LDLR-/-/15LO mice were comparable to those obtained in transgenic 5.9mPPET-15-LO mice.33 Although its activity and expression in vascular tissues were very high, nonvascular tissues showed minimal or no activity of 15-LO (Table 1
). High
activity of 15-LO, measured as 15-HETE production from
arachidonic acid ex vivo, was found in heart extracts
of LDLR-/-/15LO mice fed
chow and high-fat high-cholesterol diets, with a ratio of 4
to 10 of 15-HETE to 12-HETE (Figure 1).
LO activity in heart extracts of
LDLR-/- mice was 
8
times lower, with a 15-HETE to 12-HETE ratio of 0.12, indicating an
activity of its wild-type 12-LO–like 15-LO activity. In contrast to
the heart extract, no activity of 15-LO was detected in isolated
peritoneal macrophages of both study groups (Figure 1).
The only LO activity in the macrophages was that of the
endogenous 12-LO–like 15-LO, as indicated by the 15-HETE
to 12-HETE ratio of 0.3. Immunohistochemistry with anti-human 15-LO
antibodies revealed abundant human 15-LO expression in the
atherosclerotic lesion in the double-transgenic mice (Figure 2).
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Plasma Lipid Levels
Plasma cholesterol and triglyceride levels
were similar in LDLR-/-
and LDLR-/-/15LO mice
throughout the experiment. Although lower levels were detected in the
double-transgenic group, a sharp increase in cholesterol
levels was seen in both groups at 3 weeks of
high-cholesterol high-fat diet (Table 2).
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Susceptibility of Lipoproteins to Oxidation Ex Vivo
Because 15-LO has been suggested to oxidize lipoproteins in vivo,
we measured the susceptibility of lipoproteins isolated from
LDLR-/- and
LDLR-/-/15LO mice to
copper-induced ex vivo oxidation. Lipoprotein fraction (1.019 to 1.063)
was isolated by ultracentrifugation, and susceptibility
to copper-induced oxidation was measured by conjugated diene formation
at 234 nm. The susceptibility to oxidation of lipoproteins isolated
from both groups of chow-fed mice was similar (data not shown).
However, after 3 weeks of a high-fat high-cholesterol diet,
lipoproteins isolated from 15-LO mice were significantly
(P<0.05) more susceptible to oxidation, as measured by the
shorter lag phase in the conjugated diene formation kinetics, 87
minutes compared with 112 minutes in
LDLR-/- mice (Figure 3).
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indicates LDLR-/- mice; 
,
LDLR-/-/15LO mice.
Anti-OxLDL Antibody Levels
Levels of anti-oxLDL antibodies were slightly increased after
short-term feeding of the high-fat diet in all experimental groups.
However, no differences were evident between the
LDLR-/- and
LDLR-/-/15LO mice with
respect to the levels of anti-oxLDL antibodies throughout the study
(mean optical density±SD, 0.23±0.3 in the former group
compared with 0.21±0.4 in the latter).
Atherosclerosis in
LDLR-/- and
LDLR-/-/15LO Mice
The overexpression of 15-LO in C57B6/SJL mice did not induce
significantly more atherosclerosis than found in the
wild-type C57B6/SJL; therefore, we performed 2 experiments to assay the
effect of 15-LO overexpression on atherogenesis in the
double-transgenic mice. Forty-five 3-month-old mice in each group were
studied. The first experiment lasted for 6 weeks, and the second lasted
for 9 weeks.
Atherosclerotic lesion area was measured at the aortic sinus (Figure 4). After 3 weeks of a high-fat
high-cholesterol diet, the atherosclerotic lesion area in
the aortic sinus was significantly (P<0.001) larger in
LDLR-/-/15LO mice than in
LDLR-/- mice (107 000
versus 28 000 µm2, respectively). After 6
weeks of a high-fat high-cholesterol diet, the difference
in the atherosclerotic lesion area in the aortic sinus was slightly
smaller but significant (121 000 versus 87 000
µm2, respectively; P<0.05).
However, after 9 weeks of the atherogenic diet, the total amount of
atherosclerosis at the sinus was elevated, and no
significant difference was detected between the 2 groups of mice. At
that time, a large area of atherosclerotic lesions was detected in the
aortic arch and abdominal aorta in both mouse groups (data not shown).
It is noteworthy that at 9 weeks no differences were evident between
the experimental groups (196 000 versus 197 000
µm2, respectively; P<0.05) with
respect to the density of macrophages (macrophage
content of 32±10% in the double-transgenic mice compared with 27±8%
in the LDL-RD mice) or T lymphocytes (5±3 cells per lesion at the
aortic sinus in the double-transgenic mice compared with 7±4 cells per
lesion at the aortic sinus in the LDL-RD mice) in the sinus
atherosclerotic lesions.
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Discussion |
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The human lipid-oxidizing enzyme 15-LO has been implicated in LDL oxidation, it has been shown to be induced in atherosclerotic lesions, and its oxidation product 15-HETE is the major hydroxyeicosatetraenoic acid in the lesion.15 16 17 18 19 The present study provides evidence for its oxidative role and demonstrates that 15-LO overexpression in the vascular wall of LDLR-/- mice accelerates LDL cholesterol–mediated atherogenesis.
The role of LOs in atherogenesis has been studied in the apoE-deficient
12/15-LO knockout mice and LDL receptor knockout mice in the
present study. The 2 models develop fatty streaks as a result of
the delayed clearance of lipoproteins. Cholesterol levels
in apoE-deficient mice on chow diet reach 400 to 600 mg/dL as a result
of chylomicrons and VLDL remnant accumulation. These mice develop fatty
streaks and fibrous plaque lesions at branch points and major vessels.
LDLR-/-
mice develop mainly fatty streaks when fed a
high-fat high-cholesterol diet only.38 The
results of the present study are in accord with the recent results
obtained in apoE-deficient mice, showing that the disruption of the
12/15–LO gene diminishes
atherosclerosis.24 In contrast, studies by
Shen and colleagues31 32 have shown that overexpression of
15-LO in monocytes/macrophages in New Zealand White rabbits and
heterozygous Watanabe rabbits protects against
atherosclerosis. In these studies, the protective
effect was attributed to the platelet chemorepellant product,
13-hydroxyoctadecadienoic acid. The major differences between the
studies of Shen and colleagues and the present study are the
species and the localization of 15-LO expression. In the present
study, 15-LO driven by the preproendothelin promoter was highly
expressed in mouse endothelial cells, and no 15-LO
activity was found in isolated peritoneal macrophages or other
tissues. Moreover, 15-LO distribution in the lesion, as detected by
immunohistochemistry (Figure 2
), suggests that enzyme is highly
expressed in the lesion. In the rabbit model, 15-LO driven by a
lysozyme promoter is expressed specifically in
monocytes/macrophages, but its expression in atherosclerotic
macrophages has not been demonstrated.
Evidence indicates that 15-LO is expressed and active in endothelial cells. Weak hybridization was detected in vivo in Watanabe rabbit endothelium,15 endothelial cells can generate 15-HETE,39 and inhibitors of 15-LO inhibited LDL oxidation by endothelial cells.40 Hence, 15-LO activity in endothelial cells may play a role early in atherogenesis.
The increased susceptibility to ex vivo oxidation of lipoproteins
isolated from
LDLR-/-/15LO mice may
indicate that 15-LO overexpression in endothelial cells
of the vessel wall exposes lipoproteins in the
subendothelial space to increased oxidative stress, as
it does in vitro.6 11 12 13 Because 15-LO is cytosolic, it
has been suggested that it initiates extracellular LDL oxidation by
oxidation of cell membrane lipids and that the radical membrane
products could be transferred to LDL in the vicinity of the
endothelial cell. The finding that LDL isolated from
LDLR-/-/15LO mice fed an
atherogenic diet is more susceptible to oxidation compared with LDL
isolated from LDLR-/-
mice may indicate that 15-LO overexpression in
endothelial cells contributes to LDL oxidation in this
model. However, the lack of difference in anti-oxLDL antibodies may
suggest that there is no more oxidized LDL in the plasma of
15-LO–overexpressing mice. We speculate that the induction of 15-LO in
LDL-/-/15LO mice might
accelerate atherogenesis in this mouse model by another mechanism, such
as oxidative damage to endothelial cells. The
intracellular enzyme could oxidize fatty acid inside the cell, which
could lead to oxidation of phospholipids in the cell membrane.
Alternatively, the enzyme could oxidize phospholipids in the cell
membrane directly. 15-LO oxidation products have been suggested to
affect many steps involved in atherosclerosis:
triggering the expression of adhesion molecules,22 41 42
triggering chemotactic proteins,43 44 and affecting smooth
muscle cell migration45 and the activity of peroxisome
proliferator-activated receptor-
.46 This
increased process of atherogenesis can eventually result in enhanced
foam cell formation and accelerated atherogenesis.
In summary, in the present study, we show that overexpression of 15-LO is associated with enhanced atherogenesis in LDLR-/- mice and that LDL in these mice is more susceptible to ex vivo oxidation than is LDL isolated from LDLR-/- mice.
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Acknowledgments |
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This work was supported by Binational Science Foundation grant No. 93-00193/3
Received October 13, 1999; accepted February 22, 2000.
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