© 1997 American Heart Association, Inc.
Articles |
Atherosclerosis, Vascular Remodeling, and Impairment of Endothelium-Dependent Relaxation in Genetically Altered Hyperlipidemic Mice
From the Departments of Internal Medicine and Pharmacology (D.D.H., F.M.F.), Cardiovascular Center, University of Iowa College of Medicine, Iowa City.
Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242-1081.
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Abstract |
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Abstract We examined the vascular structure and endothelium-dependent relaxation in two genetic models of hypercholesterolemia: apolipoprotein E (apoE)-knockout mice and combined apoE/LDL receptor–double-knockout mice. Intimal area was increased markedly in proximal segments of thoracic aortas from apoE/LDL receptor–knockout mice [0.13±0.03 (mean±SE) mm2] compared with normal (C57BL/6J) mice (0.002±0.002 mm2, P<.05). Despite intimal thickening, the vascular lumen was not smaller in the aortas of apoE/LDL receptor–knockout mice (0.52±0.03 mm2) than in normal mice (0.50±0.03 mm2). In apoE-deficient mice, intimal thickening was minimal or absent, even though the concentration of plasma cholesterol was only modestly less than that in the double-knockout mouse (14.9±1.1 vs 18.0±1.2 mmol/L, respectively, P<.05). Relaxation of the aorta was examined in vitro in vascular rings precontracted with U46619. In normal mice, acetylcholine produced relaxation, which was markedly attenuated by the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 µM). Relaxation to acetylcholine and the calcium ionophore A23187 was normal in apoE-deficient mice (in which lesions were minimal) but greatly impaired in the proximal segments of thoracic aortas of apoE/LDL receptor–deficient mice, which contained atherosclerotic lesions. Vasorelaxation to nitroprusside was similar in normal and apoE-knockout mice, with modest but statistically significant impairment in atherosclerotic segments of apoE/LDL receptor–knockout mice. In distal segments of the thoracic aorta of apoE/LDL receptor–deficient mice, atherosclerotic lesions were minimal or absent, and the endothelium-dependent relaxation to acetylcholine and calcium ionophore was normal. Thus, in apoE/LDL receptor–knockout mice (a genetic model of hyperlipidemia), there is vascular remodeling with preservation of the aortic lumen despite marked intimal thickening, with impairment of endothelium-dependent relaxation to receptor- and nonreceptor-mediated agonists. Atherosclerosis may be accelerated in the apoE/LDL receptor–double-knockout mouse compared with the apoE-knockout strain alone. We speculate that other factors, such as the absence of LDL receptors, may contribute to the differences in the extent of atherosclerosis in these two models of hyperlipidemia.
Key Words: gene knockout • aorta • atherosclerosis • acetylcholine • hypercholesterolemia
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Introduction |
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Previous studies of the effects of experimental atherosclerosis on vascular function have focused primarily on diet-induced atherosclerosis.1 2 3 4 Watanabe heritable hyperlipidemic rabbits, an inbred, polygenic strain that has defective LDL receptors, are the only defined genetic model of hyperlipidemia and atherosclerosis in which vascular function has been examined.5 6 7 Recently, several murine strains have been described that are genetically susceptible to atherosclerosis and thus, provide potentially valuable models to study the vascular biology of atherosclerosis.8 Atherosclerosis in mice has many features that are characteristic of lesions found in humans.8 Murine models that lack the gene encoding apoE or a combined apoE and LDL receptor knockout develop spontaneous hypercholesterolemia and atherosclerotic lesions.9 10 11 12 13 14
Atherosclerosis impairs endothelium-dependent relaxation in diet-induced experimental animal models1 2 3 4 15 and in human arteries with atherosclerosis.16 17 18 19 It is not known whether hyperlipidemia produced by selective gene knockouts is associated with vascular dysfunction. Hence, we examined the hypothesis that endothelium-dependent relaxation is impaired in two nondietary, genetic models of hypercholesterolemia: apoE-knockout and combined apoE/LDL receptor–knockout mice. To determine whether alterations in endothelium-dependent relaxation are a diffuse abnormality produced by hypercholesterolemia per se or are confined to those vessels that contain atherosclerotic lesions, we compared responses of the proximal (atherosclerotic) and distal segments of the thoracic aorta, where intimal thickening was minimal or absent. Our goal was to examine vascular responses in genetic models of atherosclerosis. We examined vascular responses in two animal models that have different degrees of atherosclerosis.
Finally, we examined structural changes in the atherosclerotic aorta to determine whether vascular "remodeling" had occurred. In remodeling during atherosclerosis, intimal thickening is associated with outward displacement of the vessel wall so that the vascular lumen is relatively preserved.20 21 22 Vascular remodeling has been observed in diet-induced atherosclerosis in experimental animals and in coronary arteries of humans with atherosclerosis20 21 22 but has not been described in a genetic model of atherosclerosis.
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Methods |
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Animals
Procedures used in these experiments were approved by the University of Iowa Animal Care and Use Preview Committee. We studied C57BL/6J (normal), homozygous apoE–deficient, and double-homozygous apoE and LDL receptor–deficient mice that were third- or fourth-generation hybrids (with a 129xC57BL/6J background) from the colony at The Jackson Laboratory, Bar Harbor, ME. The lines were derived from brother-sister matings using knockout animals initially created by homologous recombination in embryonic stem cells. Male and female mice were fed regular mouse chow and water ad libitum. The ages (in weeks) of normal, apoE-deficient, and apoE/LDL receptor–deficient mice were 16±1, 19±3, and 19±1 and the weights (in grams) were 25±1, 30±2, and 31±1, respectively. Plasma cholesterol levels in whole plasma and fast protein liquid chromatography fractions were determined by colorimetric enzymatic assays (Boehringer Mannheim kit No. 126012) in 96-well microplates. After an overnight fast, 100 µL of mouse plasma was withdrawn, fractionated on a Superose 6 column (Pharmacia) with 10 mmol/L Tris-buffered saline (pH 8.0), pumped at 0.5 mL/min, and collected in 0.5-mL fractions as described previously.23
Vascular Ring Preparation
Studies were performed on aortic rings in vitro. On the day of
the study, the mouse was anesthetized with sodium pentobarbital
injection (0.2 mg/g body weight), and the thoracic aorta was
removed and immediately placed in Krebs' buffer of the following ionic
composition (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5,
MgSO4 1.2, KH2PO4 1.2,
NaHCO3 25, and glucose 12. Loose connective tissue in the
adventitia was removed carefully to avoid damage to the
endothelial surface, and the vessel was cut into rings
(3 to 4 mm long) from the ascending aorta and distal thoracic
aorta. Typically, four rings per mouse were studied. Aortic rings were
mounted on stainless steel hooks and suspended in vertical organ baths
containing 25 mL Krebs' solution maintained at 37°C and aerated with
a mixture of 95% O2 and 5% CO2. The rings
were connected to force transducers to measure isometric tension.
Resting tension was gradually increased to reach the final tension of
0.5 g, and the rings were allowed to equilibrate for at least 90
minutes and washed at 20-minute intervals. The rings were left at this
optimal resting tension throughout the remainder of the study.
Protocols
Aortic segments were precontracted by using a
thromboxane analogue [U46619
(9,11-dideoxy-11a,9a-epoxy-methanoprostaglandin
F2
)]. After stabilization at submaximal contraction
(~50% of maximum), responses to cumulative concentrations of
acetylcholine and nitroprusside were tested both in the presence and
absence of an inhibitor of nitric oxide synthase
L-NNA (100 µmol). Responses to cumulative concentrations
of calcium ionophore A23187 were also studied. Between each
concentration-response curve, the vessels were washed at least three
times with fresh Krebs' buffer and allowed to reequilibrate.
To evaluate a possible contribution of superoxide anion radicals to endothelial dysfunction, we studied responses to acetylcholine and nitroprusside in the presence of SOD (100 U/mL). We also studied responses to acetylcholine in the aorta from apoE/LDL receptor–deficient mice that had been injected with PEG-SOD for 5 days (50 U · g-1 · d-1).3
Drugs
Acetylcholine, sodium nitroprusside, L-NNA, SOD,
U46619, and calcium ionophore A23187 were obtained from Sigma Chemical
Co. Stock solution (1 mmol) of A23187 was prepared by dissolving the
drug in 50% DMSO and 50% isotonic saline. Stock solution of U46619
was prepared by dissolving it in ethanol, and subsequent dilutions were
made in isotonic saline. All other drugs were dissolved and diluted in
isotonic saline. All concentrations are expressed as the final
concentration of each drug in the organ bath.
Assessment of Atherosclerosis
After completion of the experiment, proximal and distal segments
of thoracic aorta were fixed in formalin, and 8- to 10-µm sections
were cut and stained with Verhoeff–Van Gieson's, and
hematoxylin/eosin stains. We obtained five rings from anatomically
constant sites: one from the midpoint of the ascending aorta (
2
mm from the aortic valve), one from the midaortic arch (midway between
the innominate and left carotid artery), and three rings from the
descending thoracic aorta (1, 4, and 7 mm from the distal to
the subclavian artery). Four of the five rings (excluding the one from
the midaortic arch) that were used for morphometry were also used for
studies of vascular reactivity. For morphometry, we obtained samples
from normal (n=17), apoE-knockout (n=15), and apoE/LDL
receptor–knockout (n=28) mice. Morphometric (quantitative)
determination of the size of the intima and media was performed with an
image analyzer as described previously.20 24 In
brief, the cross-sectional areas of the intima and media were
projected and digitized. To determine the luminal area, the
cross-sectional area enclosed by the internal elastic lamina was
corrected to a circle by applying the form factor
l2/4
to the measurement of the internal
elastic lamina, where l is the length of the lamina; the luminal area
was calculated as the difference between the corrected area within the
internal elastic lamina and the intimal area.
Statistical Analysis
All data are expressed as mean±SE; n indicates the number of
animals. Relaxation responses to acetylcholine, A23187, and sodium
nitroprusside were expressed as the percent relaxation from the amount
of precontraction produced by U46619. The response was recorded as
the cumulative relaxation response at each dose, expressed as a
percentage of the initial precontraction tension. The EC50
values were calculated by using a computer program that assumes a
linear curve between two points around 50% of the maximum response.
Comparisons were made using either a paired or unpaired Student's
t test. The Bonferroni correction was used for multiple
comparisons. Statistical significance was accepted at
P<.05.
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Results |
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The average plasma cholesterol levels in normal (C57BL/6J), apoE-deficient, and combined apoE/LDL receptor–deficient mice were 2.8±0.2 mmol/L (n=17), 14.9±1.1 mmol/L (n=26) (P<.05 versus normal), and 18.0±1.2 mmol/L (n=26) (P<0.05 versus normal and apoE deficient), respectively. Fig 1
shows the cholesterol profile for normal, apoE-deficient,
and apoE/LDL receptor–deficient mice. As expected from previously
published studies,12 normal mice do not have prominent
cholesterol-containing particles in the VLDL and LDL
density ranges. Their most prominent peak is in the HDL range near
fraction 35. In contrast, apoE-deficient mice have a prominent peak in
the largest particles near fraction 20. ApoE/LDL receptor–deficient
mice have an additional large peak in the LDL range around fraction
28.
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),
apoE-knockout (
), and combined apoE/LDL receptor–knockout
(
) mice that were fed a normal diet.
Morphometry and Histology
There was marked thickening of the intima-media of the proximal
(ascending) aorta of apoE/LDL receptor–deficient mice, and minimal
(size) atherosclerotic lesions of the proximal (ascending) thoracic
aorta from apoE-deficient mice (Figs 2 and 3). No
intimal thickening or lesions were observed in the aortas of normal
mice (Fig 3). Despite intimal thickening in apoE/LDL receptor–knockout
mice and marked differences in the severity of
atherosclerosis, the area of the lumen was similar in
the proximal thoracic aortas from normal, apoE-deficient, and
atherosclerotic apoE/LDL receptor–deficient mice (Fig 3).
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There were minimal or no atherosclerotic lesions in the distal segments of thoracic aortas from apoE/LDL receptor–knockout mice. The area of the vascular lumen was similar in distal segments of thoracic aortas of normal (0.38±0.02 mm2), apoE-knockout (0.42± 0.03 mm2), and apoE/LDL receptor–knockout (0.36±0.02 mm2) mice.
Responses to U46619
The optimal resting tension in all groups of mice was 0.5 g.
The maximal response to U46619 was 1.41±0.05 g in the normal aortas,
similar in aortas from apoE-deficient mice, and smaller in the proximal
segments of thoracic aortas of apoE/LDL receptor–deficient mice
(0.92±0.08 g, P<.05). The EC50 for contraction
with U46619 was -7.74±0.06 in normal mice and was not significantly
different in vessels from apoE-deficient or apoE/LDL
receptor–deficient mice.
Responses in Normal C57BL/6J Mice
In vessels from normal mice, acetylcholine produced
concentration-dependent relaxation (Fig 4), and the response was
similar in both proximal and distal segments of the thoracic aorta (Fig 5). Relaxation to acetylcholine was
similar in female and male mice (data not shown). Relaxation to A23187
and nitroprusside was also similar in proximal and distal segments of
thoracic aortas of normal mice (Fig 5).
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In normal mice, relaxation to acetylcholine was markedly attenuated by L-NNA (100 µmol), a nitric oxide synthase inhibitor (P<.05). Maximum relaxation in response to acetylcholine was 76±5% and 38±4% in the absence and presence of L-NNA, respectively. L-NNA did not inhibit but instead enhanced (produced a leftward shift of the dose-response curve) responses to sodium nitroprusside in the normal mouse aorta (P<.05). The EC50 for nitroprusside was -7.62±0.13 and -8.24±0.10 in the absence and presence of L-NNA, respectively.
Responses in ApoE-Knockout Mice
In apoE-knockout mice, relaxation in response to acetylcholine and
nitroprusside in the proximal segments of the thoracic aorta, which had
minimal or no intimal thickening, was similar to that of the distal
segments of the thoracic aorta (Fig 6).
Relaxation was similar to that in normal mice (Fig 5
). The
EC50 for relaxation with acetylcholine and nitroprusside
was similar in the aortas of normal and apoE-deficient mice
(Table).
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Relaxation to acetylcholine was markedly attenuated by L-NNA in apoE-deficient mice (data not shown). Thus, vasorelaxation in response to acetylcholine was mediated by nitric oxide in apoE-knockout mice as well as in normal mice. Similar to its effects on the normal aorta, L-NNA enhanced the response to sodium nitroprusside in aortas of apoE-knockout mice (data not shown).
Responses in ApoE/LDL Receptor–Double-Knockout Mice
In apoE/LDL receptor–mutant mice, relaxation to acetylcholine was
markedly impaired in the proximal segments of the thoracic aorta, which
had atherosclerotic lesions when compared with the distal segments of
thoracic aorta, which had minimal or no intimal thickening (Figs 4 and 7). In response to acetylcholine,
relaxation of proximal segments of thoracic aortas of apoE/LDL
receptor–knockout mice was significantly impaired when compared with
proximal segments of thoracic aortas of either normal or apoE-knockout
mice (Figs 4 through 6). Impairment of relaxation to acetylcholine in
the proximal thoracic aorta of apoE/LDL receptor–knockout mice was
similar in female and male mice (data not shown). Relaxation of the
proximal (atherosclerotic) segments of the thoracic aorta in response
to A23187 was markedly impaired compared with distal segments of the
thoracic aorta of apoE/LDL receptor–deficient mice or normal mice (Fig 7).
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10-7
mol/L. Relaxation of the proximal segments of thoracic aortas in
response to A23187 (middle) was significantly less (P<.05)
than that of the distal segments of thoracic aorta at
3x10-7 and 10-6 mmol/L A23187.
Relaxation of the proximal segments of thoracic aorta in response to
nitroprusside (right) was significantly less (P<.05) than
that of the distal segments of thoracic aorta at concentrations of
nitroprusside
In the proximal (atherosclerotic) segments of thoracic aortas of
apoE/LDL receptor–deficient mice, relaxation in response to sodium
nitroprusside was attenuated (Fig 7). Relaxation to sodium
nitroprusside in distal segments of thoracic aortas of apoE/LDL
receptor–knockout mice was similar to that of normal and
apoE-deficient mice. The EC50 for relaxation in response to
sodium nitroprusside was similar in all three groups of mice (data not
shown).
As in normal and apoE-deficient mice, relaxation of the distal segments of thoracic aortas in response to acetylcholine was markedly attenuated in the presence of L-NNA in apoE/LDL receptor–knockout mice. L-NNA did not inhibit but enhanced (produced a leftward shift of the dose-response curve) responses to sodium nitroprusside in both normal and atherosclerotic segments of thoracic aortas (data not shown).
Effect of SOD
In vitro incubation of the atherosclerotic segments of thoracic
aortas from apoE/LDL receptor–deficient mice with SOD (100 U/mL) for
30 minutes did not significantly influence
endothelium-dependent relaxation (maximum relaxation in
response to acetylcholine, 33±7%). In apoE/LDL receptor–deficient
mice (n=3) injected with PEG-SOD (for 5 days), relaxation of the
proximal atherosclerotic segments of the thoracic aorta in response to
acetylcholine (maximum relaxation in response to acetylcholine,
12±7%) was not improved.
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Discussion |
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Endothelium-dependent relaxation is impaired by atherosclerosis in humans and in several animal models of atherosclerosis.15 This is the first study to examine vascular function in hyperlipidemic mice with well-defined genetic defects. The major new findings in this study are that in the presence of atherosclerotic lesions, there is vascular remodeling and impairment of relaxation to receptor- and nonreceptor-mediated endothelium-dependent agonists (acetylcholine and A23187). The defect in endothelium-dependent relaxation observed in the proximal atherosclerotic segments of thoracic aortas of apoE/LDL receptor–knockout mice apparently was not produced by hypercholesterolemia per se, because the distal segments of the thoracic aorta (which had minimal or no intimal thickening) of the same mouse relaxed normally in response to both acetylcholine and A23187.
Endothelium-Dependent Relaxation
There have been few studies that have examined
endothelium-dependent relaxation of mouse vessels in
vitro. In normal mice, we found that acetylcholine produced relaxation
that was markedly attenuated by the nitric oxide synthase
inhibitor L-NNA. Thus, relaxation of the normal
mouse aorta in response to acetylcholine is mediated in large part by
nitric oxide.
Mechanisms that produce impaired endothelial function in atherosclerosis are not completely clear. One possibility is that smaller quantities of nitric oxide are produced by atherosclerotic vessels in response to endothelium-dependent stimuli. This seems unlikely, because levels of mRNA and protein for the endothelial isoform of nitric oxide synthase (NOS III) and the production of nitric oxide in the atherosclerotic rabbit aorta appear to be increased rather than decreased.25 26 We found that vasorelaxation in response to both acetylcholine and A23187 was impaired in the proximal aortas of apoE/LDL receptor–deficient mice, which contained atherosclerotic lesions. Impairment of responses to A23187 suggests that endothelial dysfunction extends beyond muscarinic receptors in this genetic model.
A second possible mechanism of impairment of endothelial function relates to degradation and/or inactivation of nitric oxide by superoxide anion. Incorporation of lipids within the endothelium, an early manifestation of atherosclerosis, and associated oxidative processes may be responsible for the degradation of nitric oxide. For example, increased production of superoxide anion in atherosclerotic arteries has been detected in several studies.4 27 28 Nitric oxide is rapidly inactivated by superoxide anion.29 30 The reaction of nitric oxide and superoxide anion occurs even more rapidly than the reaction of superoxide anion with SOD.29 30
A third possibility is impaired response of vascular muscle to nitric oxide. Relatively normal relaxation in response to sodium nitroprusside in normal and atherosclerotic vessels suggests that the guanylate cyclase/cGMP system is intact in vascular muscle. The slightly diminished relaxation of atherosclerotic segments of thoracic aortas from apoE/LDL receptor–deficient mice in response to sodium nitroprusside may be attributed to the severity of the lesion.
Some3 5 31 but not all32 33 studies have shown that impaired endothelium-dependent responses during hypercholesterolemia and/or atherosclerosis can be restored toward normal by SOD. In the present study, endothelium-dependent relaxation of the proximal atherosclerotic segments of thoracic aortas of apoE/LDL receptor–deficient mice was not improved by acute or chronic (5-day) treatment with SOD. We cannot exclude the possibility that the dose or duration of SOD administration was not sufficient. Nevertheless, this finding is similar to that in a recent study in patients32 and suggests that either generation of superoxide anion is not important in this model or that exogenously administered SOD may be unable to reach the site of formation of superoxide anion. Because of the extremely efficient reaction of superoxide anion and nitric oxide,29 30 SOD needs to be present at the site of superoxide radical generation to protect nitric oxide.30 Thus, our studies do not exclude a role for enhanced generation of superoxide anion as a mediator of vascular dysfunction during atherosclerosis.
We found that responses to acetylcholine did not differ in female and male mice. This finding in normal and atherosclerotic mice is different from previous reports in other species.34 35 36 37 38 It is possible that our observation is an artifact, because the number of females and males in each group was small. It is possible, however, that the finding is accurate for mice. In monkeys and rabbits, estrogen modulates vasomotion of atherosclerotic coronary arteries.35 36 Other studies indicate that in C57BL/6J mice, males are more resistant than females to hypercholesterolemia-induced atherosclerosis.39 A recent study demonstrated that basal release of endothelium-derived nitric oxide was significantly greater in the aortas of male (C57BL/6J) than in female mice.40 Thus, findings in previous studies39 40 and the present study may suggest that increased susceptibility to atherosclerosis in males, which is characteristic of several species, is not observed in C57BL/6J mice.
Severity of Atherosclerosis
A surprising finding in the present study is that intimal
thickening in the proximal segments of the thoracic aorta was much
greater in apoE/LDL receptor–knockout mice than in apoE-knockout mice.
We used mice that were <20 weeks old, and our findings with
apoE-knockout mice are consistent with previous studies, which
observed only modest atherosclerosis in apoE-knockout
mice that were <20 weeks of age.9 10 In older
apoE-knockout mice fed a normal diet, extensive
atherosclerosis develops in the proximal segments of
the thoracic aorta.9 10 11 To our knowledge, similar studies
have not been performed previously in combined apoE/LDL
receptor–knockout mice. Plasma cholesterol levels were
only modestly greater in apoE/LDL receptor–knockout mice (18.0
mmol/L) than in apoE-knockout mice (14.9 mmol/L),
which is consistent with previous studies by Ishibashi et
al.12 Thus, it was surprising that intimal thickening was
12-fold greater in the double-knockout than the apoE-knockout
mice.
A possible explanation for the marked difference in development of atherosclerosis in apoE/LDL receptor–knockout mice compared with apoE-knockout mice is the presence of a prominent population of cholesterol-rich LDL in apoE/LDL receptor–knockout mice. Larger cholesterol-rich particles, which are known to resemble ß-VLDL, predominate in both hyperlipidemic models. However, LDLs are smaller than ß-VLDLs and consequently are more likely to penetrate the endothelium and deposit in the arterial wall. Another possibility is that the absence per se of LDL receptors in the vessels is atherogenic. The role of vascular LDL receptors in atherosclerosis is unclear. LDL catabolism by the intima has been shown to be predominant.34
The observation of vascular remodeling in atherosclerotic apoE/LDL receptor–knockout mice is of interest. Several pathological processes in the intima-media of atherosclerotic arteries result in thickening of the vessel wall. In remodeling, the atherosclerotic vessel wall thickens, but there is outward displacement of the vessel wall so that the lumen is preserved. The area circumscribed by the internal elastic lamina increases as the area of the plaque contained within the lumen increases. In studies of atherosclerosis in nonhuman primates and in atherosclerotic human coronary arteries, maintenance of lumen size has been noted despite massive intimal thickening.20 21 22 Vascular remodeling is thought to require synthesis and reorganization of collagen and the extracellular matrix. Our observations indicate that the apoE/LDL receptor–knockout mouse may be a useful model for future studies of this process.
In summary, the present study shows that atherosclerosis, which is more severe in combined apoE/LDL receptor–knockout mice than in apoE-knockout mice, is associated with vascular remodeling and significant attenuation of the endothelial regulation of vascular tone. These studies of endothelial function in mice provide the foundation for additional studies of vascular biology in novel, genetically altered models.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-14388 (to D.D.H.), HL-38901 (to F.M.F.), HL-16066 and AG-10269 (to D.D.H.)., HL-39050 (to K.G.L.), and HL-49264 (to D.A.C.) and by research funds from the Veterans Administration. Kathryn G. Lamping and Frank M. Faraci are Established Investigators of the American Heart Association. We thank Kristen Rummelhart for technical assistance.
Received November 4, 1996; accepted June 16, 1997.
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