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Originally published In Press as doi:10.1194/jlr.M800009-JLR200 on February 10, 2008

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Journal of Lipid Research, Vol. 49, 1048-1055, May 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

ApoE2-associated hypertriglyceridemia is ameliorated by increased levels of apoA-V but unaffected by apoC-III deficiency*

Gery Gerritsen*, Caroline C. van der Hoogt{dagger},§, Frank G. Schaap**, Peter J. Voshol{dagger},§, Kyriakos E. Kypreos*,{dagger}{dagger}, Nobuyo Maeda§§, Albert K. Groen**, Louis M. Havekes{dagger},§,***, Patrick C. N. Rensen{dagger},§ and Ko Willems van Dijk1,*,§

* Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
{dagger} TNO-Quality of Life, Gaubius Laboratory, Leiden, The Netherlands
§ Department of General Internal Medicine, Endocrinology, and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands
** Amsterdam Medical Center Liver Center, Amsterdam, The Netherlands
{dagger}{dagger} Boston University School of Medicine, Boston, MA
§§ Department of Pathology, University of North Carolina, Chapel Hill, NC
*** Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands

* This research was conducted in the framework of the Leiden Center for Cardiovascular Research Leiden University Medical Center-TNO and supported by the Netherlands Heart Foundation (Project 2000.099), the Leiden University Medical Center (Gisela Thier Fellowship to P.C.N.R.), the Netherlands Organization for Scientific Research (NWO VIDI Grant 917.36.351 to P.C.N.R. and Netherlands Organization for Scientific Research Program Grant 903-39-291 to L.M.H.), the American Heart Association (Grant SDG 0535443T to K.E.K.), and the National Institutes of Health (Grant HL-42630 to N.M.). Back

Published, JLR Papers in Press, February 10, 2008.

1 To whom correspondence should be addressed. e-mail: kowvd{at}lumc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.

Supplementary key words apolipoprotein E2 • apolipoprotein A-V • apolipoprotein C-III • APOE2-knockin mice • lipoprotein lipase-mediated very low density lipoprotein-triglyceride hydrolysis • adenovirus-mediated gene transfer

Abbreviations: apoE2, apolipoprotein E2; FPLC, fast-protein liquid chromatography; HSPG, heparan sulfate proteoglycan; TC, total cholesterol; TG, triglyceride


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by increased plasma levels of chylomicron and VLDL remnants and is associated with xanthomatosis and premature atherosclerosis (1). ApoE2 has a single amino acid substitution (arginine158->cysteine) compared with the common apoE3 variant, resulting in a low binding affinity for the LDL receptor (2, 3). In vivo, this is associated with impaired hepatic clearance of VLDL and chylomicron remnant particles (4), resulting in increased plasma triglyceride (TG) and total cholesterol (TC) levels. Simultaneously, apoE2 accumulates in plasma, leading to an increase in apoE-mediated inhibition of LPL-mediated TG hydrolysis (5). It has been postulated that both impaired remnant clearance and impaired remnant generation via lipolysis contribute to the hyperlipidemia associated with apoE2 (5).

We and others have found that VLDL obtained from hyperlipidemic patients homozygous for APOE2 is a relatively poor substrate for LPL-mediated lipolysis (6). Two potent modifiers of LPL activity have been described, apoA-V and apoC-III, that are encoded in the same gene cluster on chromosome 11 (7). In vitro and in vivo mouse studies indicate that apoA-V stimulates LPL-mediated TG hydrolysis and that apoC-III inhibits this process (8–12). Overexpression of apoA-V in mice reduces plasma TG levels via the stimulation of LPL activity (13), and overexpression of apoC-III results in increased plasma TG levels via the inhibition of LPL (14). Studies in Apoc3-knockout mice show accelerated LPL-mediated TG hydrolysis (15, 16). Deficiency in apoA-V in both mice and humans is associated with hypertriglyceridemia (17–19).

In the present study, we investigated the role of LPL-mediated TG hydrolysis in apoE2-associated hyperlipidemia in vivo. A direct increase of active LPL in the circulation of APOE2-knockin (APOE2) mice via heparin injection and via adenovirus-mediated gene transfer of LPL reduced both TG and TC levels. Indirect stimulation of the LPL activity via the deletion of endogenous Apoc3 did not affect the plasma TG levels, whereas indirect stimulation via adenovirus-mediated overexpression of Apoa5 resulted in decreased plasma TG and TC levels. Thus, stimulation of LPL activity via apoA-V overexpression or apoC-III deficiency occurs via different mechanisms.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Adenoviral constructs
The adenoviral vectors expressing enzymatically active LPL (AdLPL) and inactive LPL (AdLPL-inactive) were kindly provided by Dr. S. Santamarina-Fojo (20). The generation of the adenoviral vectors expressing apoA-V (AdApoa5), the control empty vector (AdEmpty), and β-galactosidase (AdlacZ) has been described (8, 13). Expansion, purification, and titration of the adenoviral vectors were performed as described previously (21). Before in vivo administration, the adenoviral vectors were diluted to a dose of 5 x 108 plaque-forming units (pfu) in 200 µl of sterile PBS.

Mouse models
APOE2-knockin mice, carrying the human APOE2 gene in place of the mouse Apoe gene, have been described (22). These mice were backcrossed eight times with C57BL/6 mice to achieve a more homogenous genetic background and subsequently intercrossed to obtain homozygous APOE2 mice. Apoc3–/– mice were obtained from Jackson Laboratories (Bar Harbor, ME) and intercrossed with APOE2 mice to obtain APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice. The mice were fed a regular mouse diet (SRM-A; Hope Farms, Woerden, The Netherlands) and given free access to food and water. At least 5 days before adenovirus injection, mice were transferred to filter-top cages in designated rooms. All animal experimentation protocols were approved by the Committee on Animal Experimentation of the Leiden University Medical Center.

Adenovirus-mediated gene transfer in mice
Male APOE2 mice at 13–18 weeks of age were selected for injection with AdLPL or AdLPL-inactive. A dose of 5 x 108 pfu adenovirus was injected into the tail vein. Before and 5 days after the administration of adenovirus expressing active LPL or inactive LPL, mice were fasted for 4 h and a blood sample for the determination of lipids was collected by tail bleeding, using diethyl-p-nitrophenyl phosphate (paraoxon; Sigma)-coated heparinized capillary tubes (Hawksley, Sussex, England).

Female APOE2 mice at 13–18 weeks of age were injected with a dose of 5 x 108 pfu AdApoa5 or AdEmpty. Three hours before this virus injection, the mice were injected with 5 x 108 pfu AdlacZ to saturate the uptake of viral particles by hepatic Kupffer cells (23). Before and 4 days after virus injection, mice were fasted for 4 h and a blood sample for the determination of lipids was collected in paraoxon-coated capillaries by tail bleeding.

Lipid determinations
Plasma was isolated from blood samples obtained from the mice by centrifugation. TG and TC levels were measured enzymatically (Sigma). Human apoE levels were measured by sandwich ELISA as described previously (24). The circulating human apoE level in homozygous APOE2-carrying mice was 3.1 ± 0.9 mg/dl.

Lipoproteins were separated using fast-protein liquid chromatography (FPLC). Plasma pools obtained from the various groups of mice were diluted five times using PBS. A volume of 50 µl was injected onto a Superose 6 column (3.2 x 30 mm, AKTA system; Pharmacia, Uppsala, Sweden) to separate lipoprotein fractions. Elution fractions of 50 µl were collected and assayed enzymatically for TG and TC levels as described above.

Heparin treatment
Heparin (or vehicle) was administered to APOE2 mice after a period of 4 h of fasting via intravenous injection of a dose of 0.5 U/g body weight. Blood samples of ~30 µl were drawn via the tail vein at 0, 10, 30, 60, and 120 min after heparin injection using paraoxon-coated capillaries. Plasma TG levels were measured enzymatically as described above.

Fat load
The fat load response was determined in male APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice aged 13–20 weeks. The mice were fasted overnight and given an intragastric olive oil load (Carbonell, Cordoba, Spain) of 400 µl. Before the olive oil load and 3 h after the load, a blood sample was drawn via the tail vein for plasma TG determination. The circulating TG levels were corrected for the TG level before the fat load.

Statistical analysis
Data were analyzed using the nonparametric Mann-Whitney test. P < 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of increased plasma LPL level on lipid levels in APOE2 mice
Intravenous injection of heparin results in an increase of active LPL in the circulation. Stimulation of LPL activity in APOE2 mice via injection of heparin (0.5 U/g body weight) reduced the hyperlipidemia in a time-dependent manner (Fig. 1 ). The maximum reduction was observed at 60 min after injection. At this time point, the plasma TG levels decreased by 53% (Fig. 1A) and the TC levels decreased by 18% (Fig. 1B). This effect is similar to the effect of heparin as observed in wild-type mice (Fig. 1).


Figure 1
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  		Fig. 1. Plasma lipid levels of apolipoprotein E2 (APOE2) mice after heparin treatment. Fasted APOE2 mice were injected with heparin. Before (time 0) and 10, 30, 60, and 120 min after injection, plasma samples were obtained and assayed for triglyceride (TG; A) and cholesterol (B). The values are represented as percentages of preinjection levels and are means ± SD for n = 4 mice per group. WT, wild type.

 
APOE2 mice were injected with adenovirus expressing enzymatically active and inactive LPL to further investigate the suitability of apoE2-containing particles as substrate for LPL (Fig. 2 ). At day 5 after injection of 5 x 108 pfu AdLPL-inactive, APOE2 mice exhibited a 129% increase in plasma TG levels (Fig. 2A), whereas TC levels were hardly affected (+12.5%) (Fig. 2B). The increase in plasma TG after AdLPL-inactive administration may be a nonspecific virus-mediated effect but may also result from the opsonization of lipoprotein with inactive protein, thereby preventing the interaction with active LPL at the endothelial surface. In contrast, at day 5 after injection of 5 x 108 pfu AdLPL, APOE2 mice exhibited an 85% decrease in plasma TG levels (Fig. 2A) and a 40% decrease in TC levels (Fig. 2B). The lipoprotein distribution as determined by FPLC showed a decrease in VLDL-TG and VLDL-TC to wild-type levels after injection of AdLPL, indicating an accelerated conversion of apoE2-containing VLDL particles by overexpression of LPL (Fig. 3 ).


Figure 2
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  		Fig. 2. Plasma lipid levels of APOE2 mice injected with adenoviral vectors expressing enzymatically active LPL (AdLPL) and inactive LPL (AdLPL-inactive). APOE2 mice were injected with 5 x 108 plaque-forming units (pfu) AdLPL-inactive or AdLPL. Before (open bars) and at day 5 after adenovirus injection (closed bars), fasted plasma samples were assayed for TG (A) and cholesterol (B). Values are represented as means ± SD for n = 3 mice per group.

 

Figure 3
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  		Fig. 3. Lipoprotein profiles of APOE2 mice injected with AdLPL-inactive and AdLPL. Pooled plasma samples of APOE2 mice injected with 5 x 108 pfu AdMock, AdLPL-inactive, and AdLPL (n = 3 per group) were subjected to fast-protein liquid chromatography (FPLC), and the elution fractions were assayed for TG (A) and cholesterol (B). IDL, intermediate density lipoprotein.

 
Effect of apoC-III deficiency on lipid levels in APOE2 mice
The main endogenous inhibitor of LPL, apoC-III, was deleted from the genetic background of APOE2 mice by crossbreeding with Apoc3-knockout mice. The effect of Apoc3 deficiency on APOE2-associated hyperlipidemia was investigated in APOE2 mice heterozygous or homozygous deficient for the endogenous Apoc3 gene (Fig. 4 ). Surprisingly, the plasma TG levels were not different between APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice (Fig. 4A). The TC levels in APOE2 mice showed a tendency toward a decrease by Apoc3 deficiency (Fig. 4B). No differences in plasma lipid levels were found between male and female mice (data not shown). The distribution of TG and TC over the lipoprotein fractions was measured after separation via FPLC (Fig. 5 ). No differences were observed between APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice in TG distribution (Fig. 5A), which is in full accordance with the total plasma TG levels, However, the tendency toward a decrease in TC levels by Apoc3 deficiency is reflected by a decrease in cholesterol in the VLDL/LDL fractions in the APOE2.Apoc3+/– and APOE2.Apoc3–/– mice (Fig. 5B).


Figure 4
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  		Fig. 4. Plasma lipid levels of APOE2 mice deficient for Apoc3. Fasted plasma samples were obtained from APOE2 mice (open bars), APOE2.Apoc3+/– mice (hatched bars), and APOE2.Apoc3–/– mice (closed bars). The samples were assayed for TG (A) and cholesterol (B). Values are represented as means ± SD for n = 5 mice per group.

 

Figure 5
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  		Fig. 5. Lipoprotein profiles of APOE2 mice deficient for Apoc3. Pooled plasma samples of APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice (n = 5 per group) were subjected to FPLC, and the elution fractions were assayed for TG (A) and cholesterol (B).

 
To further analyze the effect of Apoc3 deficiency in APOE2 mice on TG metabolism, mice were given an intragastric olive oil load. The increase in plasma TG levels was measured over a period of 3 h and the TG appearance rate was determined. The increase in TG level over 3 h was not different between APOE2 mice (0.9 ± 1.6 mM; n = 5), APOE2.Apoc3+/– mice (1.1 ± 0.4 mM; n = 5), and APOE2.Apoc3–/– mice (1.2 ± 1.5 mM; n = 4).

Effect of adenovirus-mediated expression of Apoa5 on lipid levels in APOE2 mice
The activator of LPL, apoA-V, was expressed in APOE2 mice via a recombinant adenoviral vector (Fig. 6 ). Injection of a moderate dose of AdApoa5 (5 x 108 pfu) reduced plasma TG by 81% (P < 0.05) and TC by 41% (P < 0.05) compared with AdEmpty. Analysis of lipoprotein fractions separated by FPLC revealed that the apoA-V-mediated reduction of plasma TG was associated with a 4-fold reduction in VLDL-TG, whereas the TG level in the intermediate density lipoprotein/LDL fraction was affected to a minor degree. The reduction in plasma TC level was associated with a 2-fold reduced VLDL-TC level (Fig. 7 ).


Figure 6
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  		Fig. 6. Plasma lipid levels of APOE2 mice injected with AdApoa5. APOE2 mice were injected consecutively with AdlacZ (5 x 108 pfu) and AdApoa5 or AdEmpty (5 x 108 pfu). Before injection (open bars) and at 4 days after injection (closed bars), fasted plasma was collected from the individual mice and assayed for TG (A) and cholesterol (B). Values are represented as means ± SD for five mice per group. * P < 0.05.

 

Figure 7
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  		Fig. 7. Lipoprotein profiles of APOE2 mice injected with AdApoa5. Pooled plasma samples of APOE2 mice injected with 5 x 108 pfu AdMock or AdApoa5 (n = 5 per group) were subjected to FPLC, and the elution fractions were assayed for TG (A) and cholesterol (B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we have addressed the hypothesis that alleviating the apoE2-mediated inhibition of lipolysis can reduce the apoE2-associated hypertriglyceridemia. Using the APOE2 mouse model, we first increased circulating LPL activity directly via heparin injection. This resulted in a reduction of the TG and TC levels in APOE2 mice (Fig. 1). Likewise, injection of adenovirus expressing LPL in APOE2 mice reduced the plasma TG and TC levels (Fig. 2). The reduction in TG and TC was confined mainly to the VLDL-sized fractions (data not shown). These experiments demonstrated that apoE2-containing particles are a suitable substrate for LPL. Subsequently, LPL was stimulated indirectly via its oppositely acting modulators apoC-III and apoA-V. Apoa5 overexpression did reduce the APOE2-associated hypertriglyceridemia in APOE2-knockin mice (Fig. 6). In contrast, the APOE2-associated hypertriglyceridemia was not affected by Apoc3 deficiency (Fig. 4). Our data indicate that a direct increase of LPL activity by increasing circulating LPL levels reduces APOE2-associated hypertriglyceridemia. The indirect stimulation of LPL activity via Apoa5 overexpression but not Apoc3 deficiency ameliorates the APOE2-associated hypertriglyceridemia. We conclude that apoA-V is apparently dominant over apoC-III in the improvement of APOE2-associated hypertriglyceridemia. Moreover, apoA-V and apoC-III modulate LPL activity via distinct mechanisms.

The effects of AdApoa5 injection on plasma lipid levels of wild-type mice have been reported by us previously (8). At the dose used in the current study (5 x 108 pfu/mouse), AdApoa5 resulted in a significant decrease in TC (68%) and TG (65%) in wild-type mice. In APOE2 mice, AdApoa5 decreased TG more severely (81%) compared with TC (41%), in agreement with a primary effect of apoA-V on lipolysis. The effect of apoC-III deficiency on plasma lipid levels of wild-type mice revealed a more dramatic effect on TG levels (74% reduction) compared with TC levels (56% reduction) (15). This is also in agreement with an increased activity of LPL in the absence of apoC-III (11, 12, 15). Compared with wild-type mice, the APOE2 mice displayed a combined hyperlipidemia, characterized by increased levels of both TC and TG. Because increasing circulating LPL levels decreased the APOE2-associated hyperlipidemia, and specifically the hypertriglyceridemia, the identical plasma TG levels of APOE2, APOE2.Apoc3+/–, and APOE2.Apoc3–/– mice are a strong indication that apoC-III deficiency truly has very little effect on the APOE2-associated hypertriglyceridemia. Interestingly, the APOE2-associated hypercholesterolemia seems reduced by Apoc3 deficiency (Fig. 4). It has been reported that, besides inhibiting LPL, apoC-III can also directly inhibit the hepatic uptake of apoE-containing lipoproteins by the liver (25). Apparently, Apoc3 deficiency in APOE2 mice does not affect LPL activity, as is evident from unaltered plasma TG levels, but may enhance the removal of cholesterol-enriched lipoprotein remnant particles, as is evident from reduced (V)LDL-cholesterol levels (Fig. 5).

Addition of apoE to lipoproteins results in a decrease in the LPL-mediated TG hydrolysis (26–28). This can at least partially explain the hypertriglyceridemia that is found in APOE2-associated familial dyslipidemia, which is characterized by plasma accumulation of apoE-enriched lipoproteins. It has been proposed that the inhibition of LPL activity is caused by displacement of the LPL coactivator apoC-II from the apoE2-rich lipoprotein particles (5). However, this is difficult to reconcile with the observation that indirect stimulation of LPL activity via apoA-V overexpression ameliorates the APOE2-associated hyperlipidemia, especially because it has been demonstrated that the LPL-activating effect of apoA-V is dependent on the presence of apoC-II (8). Thus, other mechanisms might underlie the inhibitory effect of apoE2 on LPL activity.

Under normal conditions, LPL-mediated TG hydrolysis takes place mainly at the endothelial cell surface and thus may be affected by the interaction between the TG-containing particle and the cell surface where LPL is localized. This interaction involves the association of TG-rich particles and endothelial surface-bound heparan sulfate proteoglycans (HSPGs) via apoE (29). It has been shown that apoE2 is partly defective in the association with HSPGs (30), and this could also explain part of the apoE2-associated hypertriglyceridemia. In agreement with this hypothesis, it has been found in vitro that VLDL obtained from APOE2 homozygous familial dyslipidemia patients is effectively lipolyzed by LPL in solution but is poorly lipolyzed by HSPG-bound LPL (6). Thus, apoE2-containing VLDL may be defective in the physical association with the endothelial surfaces where LPL-mediated TG hydrolysis takes place in vivo. This would explain why both additional soluble LPL via adenovirus-mediated gene transfer and increasing soluble LPL via heparin injection rescue the apoE2-associated hyperlipidemia. Intriguingly, this explanation is also in agreement with the observation that additional apoA-V rescues the apoE2-associated hyperlipidemia. It was found recently that the LPL-activating effect of apoA-V involves enhanced binding to HSPGs (10, 31). Thus, additional apoA-V on the TG-rich particle apparently overcomes the apoE2-mediated inhibition of HSPG binding. It is interesting that apoC-III deficiency cannot overcome this binding defect, despite the postulated inhibition of HSPG binding by apoC-III (14, 32). However, the in vivo contribution of HSPGs to the lipolysis of TG-rich lipoprotein particles still remains to be determined.

Apart from a stimulatory effect on LPL, it may be postulated that the decrease in plasma TG of APOE2 mice after the expression of apoA-V results from a decrease in the VLDL-TG secretion rate by the liver. We showed previously a 30% decreased VLDL-TG secretion rate after adenovirus-mediated overexpression of Apoa5 in wild-type C57BL/6 mice (8), whereas others have found no effects of apoA-V on VLDL production in either APOA5 transgenic mice (33) or Apoa5–/– mice (17). Intriguingly, in the APOE2 mice, we observed no differences in the VLDL-TG secretion rate after injection of AdApoa5 or AdEmpty (data not shown). At present, we have no explanation for these apparent discrepancies but cannot exclude the possibility that apoA-V has additional unrecognized functions.

Extrapolating our mouse model data to the human, it seems likely that variation in apoA-V level will have a pronounced effect on the expression of hypertriglyceridemia. Surprisingly, we and others have found that in humans, plasma TG and apoA-V levels are positively correlated (34–36). This would appear to be a clear discrepancy between mouse and human. However, it should be noted that the human data are derived from correlation studies, whereas the mouse data are derived from direct interventions in plasma apoA-V levels. Without a complete understanding of the role and metabolism of apoA-V, the apparent discrepancy between circulating levels of apoA-V and hypertriglyceridemia remain to be resolved.

Polymorphisms in both the APOA5 and APOC3 genes have been associated with hypertriglyceridemia (19, 37–41). Because both genes are expressed in the same gene cluster and have opposing effects on TG levels, it has been hypothesized that APOA5 and APOC3 act synergistically (7, 33). However, our current data clearly indicate that apoA-V and apoC-III affect different steps in the conversion of TG-rich lipoproteins to remnants.

Submitted on September 28, 2006
Revised on January 8, 2008 and in re-revised form January 31, 2008.


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 ABSTRACT
 INTRODUCTION
 METHODS
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 DISCUSSION
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R. M. Mansouri, E. Bauge, P. Gervois, J. Fruchart-Najib, C. Fievet, B. Staels, and J.-C. Fruchart
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