doi: 10.1161/hq1201.100221
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Arg123-Tyr166 Domain of Human ApoA-I Is Critical for HDL-Mediated Inhibition of Macrophage Homing and Early Atherosclerosis in Mice
From the Center for Experimental Surgery and Anesthesiology (P.H., K.P., A.M., P.V., R.Q., M.L., E.D., H.B.) and the Center for Molecular and Vascular Biology (G.T.), Katholieke Universiteit Leuven, Leuven, Belgium; the Joseph Stokes Jr. Research Institute (S.L.-K., M.N., M.C.P.), The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia; and INSERM U525 (D.S., E.N.), IFR 14 "Coeur Muscle et Vaisseaux" and Faculté de Médecine Pitié, Salpêtrière/Université Pierre et Marie Curie, Paris, France. Dr Theilmeier is now at Klinik und Poliklinik für Anästhesiologie und operative Intensivmedizin, University of Muenster, Muenster, Germany.
Correspondence to Paul Holvoet, PhD, Center for Experimental Surgery and Anesthesiology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail paul.holvoet{at}med.kuleuven.ac.be
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Abstract |
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Atherosclerosis was studied in apolipoprotein E (apoE) knockout mice expressing human apolipoprotein A-I (apoA-I) or an apoA-I/apolipoprotein A-II (apoA-II) chimera in which the Arg123-Tyr166 central domain of apoA-I was substituted with the Ser12-Ala75 segment of apoA-II. High density lipoprotein (HDL) cholesterol levels were identical in apoA-I and apoA-I/apoA-II mice, but at 4 months, plaques were 2.7-fold larger in the aortic root of the apoA-I/apoA-II mice (P<0.01). The macrophage–to–smooth muscle cell ratio of lesions was 2.1-fold higher in apo-I/apoA-II mice than in apoA-I mice (P<0.01). This was due to a 2.7-fold higher (P<0.001) in vivo macrophage homing in the aortic root of apoA-I/apoA-II mice. Plasma platelet-activating factor acetyl hydrolase activity was lower (P<0.01) in apoA-I/apoA-II mice, resulting in increased oxidative stress, as evidenced by the higher titer of antibodies against oxidized low density lipoprotein (P<0.01). Increased oxidative stress resulted in increased stimulation of ex vivo macrophage adhesion by apoA-I/apoA-II ß-very low density lipoprotein and decreased inhibition of ß-very low density lipoprotein–induced adhesion by HDL from apoA-I/apoA-II mice. The cellular cholesterol efflux capacity of HDL from apoA-I/apoA-II mice was very similar to that of apoA-I mice. Thus, the Arg123-Tyr166 central domain of apoA-I is critical for reducing oxidative stress, macrophage homing, and early atherosclerosis in apoE knockout mice independent of its role in HDL production and cholesterol efflux.
Key Words: apolipoprotein A-I • HDL • atherosclerosis • oxidative stress • macrophage homing
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Introduction |
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Low plasma levels of HDL and of their major component, apoA-I, correlate with an increased risk for coronary heart disease.1 ApoA-I, the major protein component of HDL, is an important determinant of the concentration of HDL in human blood.2 Expression of human apoA-I in transgenic mice resulted in increased plasma levels of small HDL particles comparable to human HDL.3 HDL cholesterol levels and human apoA-I levels were highly correlated, and dietary fat increased HDL levels in these mice by both increasing the transport rates and decreasing the fractional catabolic rates of HDL cholesterol ester and apoA-I.3–6 Expression of human apoA-I in the atherosclerosis-susceptible C57BL/6J mouse strain resulted in a 7-fold reduction of lesion areas in the aorta.7 Introduction of the human apoA-I transgene in apoE knockout (KO) mice, characterized by very high levels of atherogenic ß-VLDL and accelerated progression of complex atherosclerotic lesions,8–11 resulted in a significant protection against atherosclerosis.12,13 Compared with control mice, transgenic mice expressing mouse apoA-II had increased HDL cholesterol levels but, nevertheless, exhibited increased atherosclerotic lesion development.14 The concentration of HDL cholesterol in transgenic mice expressing human apoA-II was lower than that in control mice, probably because of the production of small HDL particles that are cleared more rapidly from the circulation, suggesting that sequence differences between human and mouse apoA-II may affect HDL metabolism.15,16 Expression of human apoA-II in human apoA-I transgenic mice had no effect on either total cholesterol or HDL cholesterol, but lesion areas were 15-fold larger in the apoA-I/apoA-II transgenic mice than in apoA-I transgenic mice on an atherosclerotic diet.17
See cover and page 1870
As yet, it is not clear which domain(s) is responsible for the opposite effects of apoA-I and apoA-II. It is also not clear whether the more potent inhibition of the progression of atherosclerosis by apoA-I–containing HDL depends on its antioxidative properties and cytokine effects, with its potential either to inhibit the infiltration of macrophages into the arterial wall or to induce reverse cholesterol transport from the atherosclerotic arterial wall to the liver.
Previously, we produced apoA-I/apoA-II chimeras in which the Arg123-Tyr166 central or the Ala190-Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II.18 Binding of the chimeras with palmitoyloleoylphosphatidylcholine was associated with a similar shift of tryptophan fluorescence, and all reconstituted HDL particles had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules per particle. Circular dichroism measurements revealed 8 
-helices per apolipoprotein molecule for all apoA-I variants. Furthermore, we have demonstrated that exchange of the central and of the carboxyl-terminal domain of apoA-I with helices of apoA-II did not affect in vivo HDL metabolism. Indeed, HDL cholesterol levels of transgenic mice expressing the central or the carboxyl-terminal domain chimera were similar to those of apoA-I transgenic mice.5,19 ApoE KO mice overexpressing the carboxyl-terminal domain chimera were as well protected against atherosclerosis as the apoA-I–overexpressing apoE KO mice were. In the present study, we have generated apoE KO mice that overexpress this apoA-I/apoA-II chimera. HDL cholesterol levels of these apoA-I/apoA-II/apoE KO mice were similar to those of apoA-I/apoE KO mice. However, early fatty streak formation was faster in apoA-I/apoA-II than in apoA-I transgenic mice. Therefore, the apoA-I/apoA-II mouse is a useful model to study the contribution of the central domain of apoA-I to different protective mechanisms of inhibition of the progression of atherosclerosis independent of HDL production. We investigated to what extent these differences were due to differences in plasma oxidative stress and inhibition of monocyte adhesion and macrophage homing and differences in the cellular cholesterol efflux capacity of HDL.
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Methods |
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Animal Experiments
Animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. Transgenic mice that express human apoA-I or the human apoA-I/apoA-II chimera were generated by zygote injection into the C57BL/6J background, as described previously.5 ApoE KO mice,9 backcrossed for 10 generations into the C57BL/6J background, were purchased from Jackson Laboratory (Bar Harbor, Me). These mice had 98.4% C57BL/6J background. Males homozygous for the transgene were mated with apoE KO females, and double-homozygous offspring (third generation) were used. Mice were fed normal chow ad libitum.
Plasma Lipoprotein, Lipid, Platelet-Activating Factor Acetyl Hydrolase, and Malondialdehyde-Modified LDL Autoantibodies
Mice were fasted overnight, and blood was drawn from the retro-orbital plexus into EDTA tubes. Plasma was obtained by centrifugation. Lipoprotein fractions were separated by gel filtration on a Superdex 200 HR column and a Superose 6 column (Pharmacia).5 Levels of phospholipids in lipoprotein fractions and triglyceride levels were determined by using an enzymatic assay (Boehringer-Mannheim). The levels of cholesterol and cholesterol esters were quantified by high-performance liquid chromatography on a Zorbax ODS reversed-phase column.19 The particle size distribution was determined by native gel electrophoresis.19
Plasma platelet-activating factor (PAF) acetyl hydrolase (AH) activity and autoantibodies against in vitro malondialdehyde-modified LDL in mice were determined as described earlier.20–22
In Vivo Macrophage Homing
Macrophages were isolated, labeled with 2-µm yellow-green fluorescent latex microspheres (Molecular Probes), and injected as described.23,24 Four days after macrophage injection, the base of the heart with the ascending aorta was collected, OCT-embedded, stored at -80°C, and cryosectioned at 7 µm. Labeled macrophages were counted in 80 serial sections per mouse, spanning 1 mm of the aortic valve region.
Ex Vivo Macrophage Adhesion
Monolayers of polyomavirus-transformed mouse endothelial cells (fEND.5)25 grown on collagen-coated glass coverslips were mounted in a parallel-plate flow chamber and superfused with peritoneal macrophages labeled with 2x105/mL bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester (Molecular Probes). Adhesion was determined on 0.9 mm2 of the coverslip, as described elsewhere.26 Endothelial cells were treated for 6 hours with ß-VLDL fractions (50 µg/mL in Hanks’ balanced salt solution [GIBCO Laboratories]/1% BSA/1.2 mmol/L Ca2+) at 37°C. HDL fractions were added at 100 µg/mL.
Cholesterol Efflux
J774 cells were used to compare the abilities of HDL samples to induce efflux of cellular cholesterol as described previously.27 Cellular cholesterol was labeled by exposing the cells to [3H]cholesterol (New England Nuclear) for 24 hours at 2 µCi per well. Acyl coenzyme A:cholesterol acyltransferase inhibitor (CP 113,818, Pfizer Pharmaceutical) in dimethyl sulfoxide was added to the medium at 2 µg/mL during the labeling and equilibration stages to ensure that the radiolabeled cholesterol incorporated into the cells was present as unesterified cholesterol.
Plaque Size and Composition
Mice were euthanized, and the hearts and aortas were fixed in 4% phosphate-buffered formaldehyde and then embedded in 25% gelatin. Frozen sections (7 µm) were prepared for morphometric and immunohistochemical analysis. The first and most proximal section to the heart was taken 
100 µm distal to the point at which the aorta becomes first rounded,28 and 12 sections per heart were analyzed. Smooth muscle cells were immunostained with a monoclonal antibody against human smooth muscle -actin (clone 1A4, Dako). Macrophages were detected with rat biotinylated monoclonal antibody against mouse Mac-3 antigen (clone M3/84, Pharmingen). Blinded analysis of positive sections immunostained for smooth muscle cells and macrophages was performed with the Quantimet600 image analyzer (Leica). A color threshold mask for immunostaining was defined to detect the red or brown color by sampling, and the same threshold was applied to all samples. The lesion area with positive color was recorded.29
Statistical Analysis
The significance of differences in lipoprotein values and in atherosclerotic lesion areas was tested by the Kruskal-Wallis test, followed by the Dunn multiple comparisons test. A value of P<0.05 was considered statistically significant.
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Results |
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Effects of ApoA-I Genotype on Lipoprotein Levels
The concentrations of non-HDL cholesterol in apoE KO mice and in mice expressing human apoA-I or the apoA-I/apoA-II chimera were not different: 580±25, 610±30, and 550±26 mg/dL, respectively. Triglyceride levels were also very similar. Expression of apoA-I and of the apoA-I/apoA-II chimera resulted in a 2.1- and 2.0-fold increase, respectively, in HDL cholesterol (Figure 1A) and a 2.9- and 2.5-fold increase, respectively, in HDL phospholipids (Figure 1B). Levels of human apoA-I and of the apoA-I/apoA-II chimera were similar: 210±11 and 180±5.8 mg/dL, respectively. Thus, the apolipoprotein-to-cholesterol-to-phospholipid ratio of HDL from apoA-I and apoA-I/apoA-II mice was very similar and was in agreement with the similar HDL distribution profiles. Indeed, consistent with our previous study,5 particles of apoA-I/apoE KO and apoA-I/apoA-II/apoE KO mice were polydisperse, with major populations of particles with diameters of 7.2, 8.4, 9.2, 9.6, and 10.2 nm, respectively. Agarose gel electrophoresis revealed that expression of either human apoA-I or apoA-I/apoA-II resulted in an increase of lipoprotein particles with
mobility (90% of total HDL) and pre-ß mobility (10%), respectively.
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Effects of ApoA-I Genotype on Atherosclerotic Lesion Size and Composition
At 4 months, atherosclerotic lesions in the aortic arch of all mouse strains were fatty streaks consisting of lipid-loaded macrophages (Figure 2A through 2C and 2E through 2G). Consistent with our previous study,28 overexpression of human apoA-I resulted in a significant reduction of atherosclerotic plaque volume compared with apoE KO mice (Figure 2D). Expression of the apoA-I/apoA-II chimera also resulted in a significant reduction of atherosclerosis. However, mean lesion volumes were significantly larger (P<0.001) in aortic segments of apoA-I/apoA-II transgenic mice than in those of apoA-I transgenic mice (Figure 2D).
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Figure 2E through 2G shows the accumulation of macrophages in fatty streaks in the aortic root of apoE KO mice, apoA-I/apoE KO mice, and apoA-I/apoA-II/apoE KO mice. The macrophage–to–smooth muscle cell ratio was 3.2-fold lower (P<0.01) in apoA-I/apoE KO mice and 1.5-fold lower (P<0.05) in apoA-I/apoA-II/apoE KO mice compared with apoE KO mice. The macrophage content was significantly higher (P<0.01) in apoA-I/apoA-II than in apoA-I transgenic mice (Figure 2H).
In Vivo Macrophage Homing and Ex Vivo Macrophage Adhesion
Previously, we have demonstrated that peritoneal macrophages display a leukocyte-typical homing pattern in vivo.24 After intravenous injection in 4-month-old apoE KO mice, 316±39 macrophages were found in the aortic root (Figure 3A). This number is consistent with our previous data.24 Expression of apoA-I resulted in a 8.1-fold reduction (P<0.001 versus apoE KO mice) of macrophage homing. Expression of the apoA-I/apoA-II chimera resulted in only a 2.1-fold reduction of macrophage homing (P<0.01 versus apoE KO mice) (Figure 3A). Macrophage homing was significantly higher (P<0.01) in apoA-I/apoA-II mice than in apoA-I mice.
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To elucidate the mechanism of the reduction of in vivo macrophage homing in apoA-I– and apoA-I/apoA-II–overexpressing apoE KO mice, ex vivo adhesion of peritoneal macrophages to fEND.5 cells was studied in a flow chamber at a shear rate of 400 s-1. Macrophages had rolled on the endothelium before adhering firmly. When fEND.5 cells were exposed to ß-VLDL of apoE mice before superfusion, 164±17 (n=6) peritoneal macrophages adhered to mouse endothelial cells. Exposure to ß-VLDL from apoA-I and apoA-I/apoA-II mice resulted in a 17-fold (P<0.01) and 4.3-fold (P<0.05), respectively, lower number of adhering macrophages (Figure 3B). HDL of apoA-I and apoA-I/apoA-II transgenic mice reduced adhesion induced by ß-VLDL of apoE KO mice 3.5-fold (P<0.001) and 2.3-fold (P<0.01), respectively, whereas HDL of apoE KO mice had no effect (Figure 3C). The effect of HDL from apoA-I/apoA-II transgenic mice was lower (P<0.05) than that of HDL from apoA-I transgenic mice (Figure 3C).
Malondialdehyde-Modified LDL Autoantibodies and PAF-AH Activity as Measures of Oxidative Stress
Titers of malondialdehyde-modified LDL autoantibodies were 9.9±0.76 (n=10) for apoE KO mice, 4.3±0.16 for apoA-I/apoE KO mice (n=8, P<0.001 versus apoE KO), and 6.8±0.50 for apoA-I/apoA-II/apoE KO mice (n=7, P<0.001 versus apoE KO and P<0.01 versus apoA-I/apoE KO). Plasma PAF-AH activity was 143±33 nmol/mL per minute in apoE KO mice (n=6), 481±14 nmol/mL per minute in apoA-I/apoE KO mice (n=6), P=0.0043), and 255±16 nmol/mL per minute in apoA-I/apoA-II/apoE KO mice (n=6, P=0.017 versus apoE KO and P=0.0022 versus apoA-I mice).
Cholesterol Efflux
Incubation of J774 mouse macrophages with isolated HDL (corresponding to 1.5% of serum HDL) from apoA-I/apoE KO and apoA-I/apoA-II/apoE KO mice resulted in 2.2- and 2.0-fold higher cholesterol efflux, respectively, than did incubation with HDL of apoE KO mice (Figure 4A). The cholesterol efflux capacities of HDL from apoA-I and apoA-I/apoA-II transgenic mice were similar. The fractional rate of cholesterol efflux, expressed as percent cholesterol efflux per 250 µg/mL phospholipid for 4 hours, was similar for HDL from apoE KO, apoA-I/apoE KO, and apoA-I/apoA-II/apoE KO mice (Figure 4B). Thus, HDL of apoE KO mice is as good an acceptor of cellular cholesterol as is HDL of apoA-I and apoA-I/apoA-II transgenic mice when present at the same phospholipid concentration.
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Discussion |
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The association among HDL cholesterol levels, cholesterol efflux from J774 macrophages, in vivo macrophage homing, and progression of atherosclerosis was studied in apoE KO mice and apoE KO mice overexpressing human apoA-I or an apoA-I/apoA-II chimera in which the central domain of apoA-I was substituted with the helical Ser12-Ala75 segment of apoA-II. Despite similar HDL cholesterol levels in mice overexpressing apoA-I or the apoA-I/apoA-II chimera, progression of early fatty streaks was faster in the aortic arch of mice expressing the chimera than in apoA-I transgenic mice.
The reverse cholesterol transport plays a central role in the antiatherogenic effect of HDL. The first step in this process is the uptake of cellular cholesterol by lipoprotein acceptors in the interstitial fluid. Therefore, the capacity of HDL isolated from the plasma of apoE KO mice overexpressing apoA-I or the apoA-I/apoA-II chimera was compared with the capacity of HDL from apoE KO mice. The total cholesterol efflux capacity of HDL was ranked as follows: HDL of apoE KO mice<<HDL of apoA-I/apoA-II/apoE KO miceHDL of apoA-I/apoE KO mice. However, fractional cholesterol efflux rates, expressed in percent cholesterol efflux per 250 µg phospholipid per hour, for the different HDL particles were very similar, consistent with the phospholipid content being a key factor in the ability of HDL to mediate cellular cholesterol efflux.30 The similar efflux to HDL from apoA-I/apoE KO and apoA-I/apoA-II/apoE KO mice indicates that the amino acid sequence of the Arg123-Tyr166 central domain of human apoA-I is not critical for the efficiency of HDL to act as cholesterol acceptors. In the present study, the 2-fold increase of HDL cholesterol levels in apoA-I/apoE KO mice was associated with an increase in cholesterol efflux potential. Thus, our data are in agreement with earlier findings in which there was a 2.5-fold increase in HDL cholesterol level, which showed that both isolated HDL and serum of human apoA-I transgenic mice induced more cholesterol efflux than did HDL or serum from control mice.31,32 It should be noted that overexpression of human apoA-I to give only an 40% rise in HDL cholesterol did not increase the cholesterol efflux capacity of mouse serum.33
Recently, we have demonstrated that overexpression of apoA-I in apoE KO mice results in a decrease of macrophage homing that is due to reduced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.24 Our findings were in agreement with previous reports showing that inhibition of intercellular adhesion molecule-1 markedly attenuated macrophage homing to atherosclerotic plaques in apoE KO mice23 and that overexpression of apoA-I Milano resulted in reduced atherosclerosis in apoE KO mice that was due to decreased macrophage infiltration.34
The new finding of the present study is that the central domain of apoA-I is crucial for preventing macrophage homing. Previously, it has been shown that apoA-II expression in transgenic mice converts HDL to proinflammatory particles that stimulate lipid hydroxyperoxide formation in the arterial wall and induce monocyte transmigration.35 Interestingly, substitution of the central domain of apoA-I with the helical pairs of apoA-II converts HDL to fewer anti-inflammatory particles. As a result of the decreased inhibition of macrophage homing in apoA-I/apoA-II/apoE KO mice, the macrophage content of early fatty streaks was higher in apoA-I/apoA-II than in apoA-I transgenic mice.
To determine the underlying mechanism of increased macrophage homing and atherosclerosis in apoA-I/apoA-II mice, we have studied macrophage–endothelial cell interaction ex vivo. The enhancing effect of ß-VLDL from apoE KO mice on macrophage adhesion was higher than that of ß-VLDL from apoA-I/apoA-II transgenic mice, which, in turn, was higher than that of ß-VLDL from apoA-I mice. We, among others, have demonstrated that enhanced oxidative stress in apoE KO mice is associated with increased accumulation in their ß-VLDL of oxidized phospholipids that are responsible for the induction of adhesion molecule expression by endothelial cells.24,29,36,37 Thus, our data show that increased macrophage adhesion and homing in apoA-I/apoA-II mice is due to loss of the antioxidative function of HDL in these mice compared with apoA-I mice.
It has been shown that HDL inhibits cytokine-induced expression of endothelial cell adhesion molecules, resulting in reduced monocyte adhesion in vitro.38 In the present study, HDL of apoA-I and apoA-I/apoA-II transgenic mice, but not of apoE KO mice, inhibited the adhesion of macrophages induced by ß-VLDL of apoE KO mice. The inhibitory effect of HDL from apoA-I/apoA-II transgenic mice was lower than that from apoA-I transgenic mice. Recently, we have shown that PAF-AH gene transfer without increasing serum HDL cholesterol concentration in apoE KO mice results in reduced oxidative stress, decreased macrophage accumulation, and atherosclerosis in the aortic arch.29 In the present study, we show that PAF-AH activity is lower in apoA-I/apoA-II mice than in apoA-I mice, suggesting that apoA-I–mediated regulation of PAF-AH activity is one possible mechanism underlying the increased atherosclerosis in apoA-I/apoA-II mice. It remains to be investigated whether lower PAF-AH activity is due to impaired interaction of PAF-AH with apoA-I/apoA-II–containing HDL and, thus, to lower PAF-AH levels or to decreased stabilization of this enzyme by apoA-I/apoA-II compared with apoA-I. Impaired interaction can be due to decreased direct interaction with the chimera compared with wild-type apoA-I or with structural changes in HDL that are due to changes in apolipoprotein sequence. However, the composition of HDL from apoA-I– and apoA-I/apoA-II–overexpressing mice was very similar. That the apolipoprotein content of HDL can be an important determinant of the stability of an HDL-associated enzyme has recently been demonstrated for paraoxonase (PON-1). It has been shown that apoA-I stabilizes the arylesterase activity of PON-1 more than does phospholipid alone, apoA-II, or apoE.39 Also, cysteine residues in the amino-terminal region of apoA-I are important for the assembly of the enzyme into nascent HDL and, thus, for enzyme activity.40 However, we did not find a correlation between PON-1 activity and human apoA-I levels in apoE KO mice overexpressing human apoA-I. HDL-associated lecithin-cholesterol acyltransferase (LCAT) is also capable of preventing accumulation of oxidized phospholipids in LDL.41 Previously, we have demonstrated that substitution of the Arg123-Tyr166 central domain of apoA-I with the helical pair of apoA-II resulted in decreased LCAT activation.18 However, we did not detect differences in plasma activity between apoA-I and apoA-I/apoA-II transgenic mice. The plasma LCAT activity was only higher in apoA-I mice in the presence of human LCAT that was induced with a human LCAT recombinant adenovirus.19
In summary, the present data show that substitution of the Arg123-Tyr166 central domain of apoA-I with the helical pair of apoA-II does not impair HDL production. Furthermore, this central domain is not crucial for cholesterol efflux to HDL, but it is required for increasing PAF-AH activity, reducing oxidative stress in plasma and, thereby, macrophage homing and atherosclerosis in apoE KO mice. The present study indicates that the role of the central domain of apoA-I in inhibition of endothelial activation is separate from that in HDL production and reverse cholesterol transport.
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Acknowledgments |
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This work was supported by the Interuniversitaire Attractiepolen Program (P4/34), by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Program G.0263.01), and by National Institutes of Health grant HL-22633. Ann Mertens, Rozenn Quarck, and Peter Verhamme are the recipients of a fellowship from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Gregor Theilmeier was a postdoctoral fellow of the Deutsche Forschungsgemeinschaft, Bonn, Germany, and is currently a member of the Interdisciplinary Center for Clinical Research at the University of Münster.
Received July 25, 2001; accepted September 19, 2001.
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