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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2337.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

GM-CSF Deficiency Reduces Macrophage PPAR- Expression and Aggravates Atherosclerosis in ApoE-Deficient Mice

Michael Ditiatkovski; Ban-Hock Toh; Alex Bobik

From the Cell Biology Laboratory (M.D., A.B.), Baker Heart Research Institute and Autoimmunity Laboratory (M.D., B.-H.T.), Department of Immunology, Monash University, AMREP, Alfred Campus, Melbourne, Victoria, Australia. Present address for B.-H.T.: Centre for Inflammatory Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.

Correspondence to Michael Ditiatkovski, Cell Biology Laboratory, Baker Heart Research Institute, PO Box 6492, St Kilda Road Central, Melbourne, Victoria, 8008, Australia. E-mail Michael.Ditiatkovski{at}baker.edu.au

	Abstract

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Objective— Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in atherosclerotic lesions but its significance for lesion development is unknown. Consequently, we investigated the significance of GM-CSF expression for development of atherosclerotic lesions in apolipoprotein E-deficient (apoE^–/–) mice.

Methods and Results— We generated apoE^–/– mice deficient in GM-CSF (apoE^–/–.GM-CSF^–/– mice), fed them a high-fat diet, and compared lesion development with apoE^–/– mice. We measured lesion size, macrophage, smooth muscle cell, and collagen accumulation at the aortic sinus, and expression of genes that regulate cholesterol transport and inflammation. No differences in serum cholesterol were found between the 2 groups. Lesion size in hyperlipidemic apoE^–/–.GM-CSF^–/– increased by 30% (P<0.05), macrophage accumulation doubled, and collagen content reduced by 15% (P<0.05); smooth muscle cell accumulation and vascularity were unaffected. Analysis of PPAR-, ABCA1, and CD36 in lesions showed reduced expression (50%, 65%, and 55%, respectively), whereas SR-A doubled. In peritoneal macrophages, PPAR- and ABCA1 expression was also reduced by 50% and 70%, respectively, as was cholesterol efflux, by 50%. In lesions, pro-inflammatory MCP-1 and tumor necrosis factor (TNF)- expression increased 2- and 3.5-fold, respectively, vascular cell adhesion molecule (VCAM)-1 expression enhanced and interleukin (IL)-1 receptor antagonist reduced by 50%.

Conclusions— GM-CSF deficiency increases atherosclerosis under hypercholesterolemic conditions, indicating antiatherogenic role for GM-CSF. We suggest this protective role is mediated by PPAR- and ABCA1, molecules that affect cholesterol homeostasis and inflammation.

We studied effect of GM-CSF deletion on atherosclerosis in apoE^–/– mice. We observed increases in lesion size, macrophage accumulation, MCP-1, TNF-, and VCAM-1; decreases in collagen content, PPAR-, ABCA1, and cholesterol efflux from macrophages. GM-CSF promotes smaller stable atherosclerotic lesions by mechanisms dependant on PPAR- and ABCA1.

Key Words: apoE^–/– mice • atherosclerotic lesions • GM-CSF deficiency • hyperlipidemia • inflammatory cytokines • PPAR-

	Introduction

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Atherosclerosis begins early in life and frequently leads to severe complications in later life with high morbidity and mortality. It is much more complex than a simple lipid storage problem, involving inflammatory mechanisms that predominate over anti-inflammatory processes.^1,2 Atherosclerotic lesions are characterized by cholesterol accumulation, immune cell infiltrates, which include macrophages, T lymphocytes, and fibrosis.³ Inflammation appears crucial in all stages of atherosclerosis, from the very early stages of lipid accumulation through progression and ultimately complications.

Recruitment, activation, survival, and proliferation of inflammatory cells in the vessel wall importantly contribute to atherosclerosis.⁴ These effects are mediated via adhesion molecules, chemokines, cytokines, and growth factors.^2,4–6

Accumulating evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) can play a key role in atherosclerosis. GM-CSF selectively regulates growth and survival of mononuclear phagocytes.⁷ Atherogenic oxidized low-density lipoproteins induce macrophage expression of GM-CSF.^8–11 Atherosclerotic lesions from humans and rabbits exhibit elevated levels of immunoreactive GM-CSF, which is expressed by endothelial cells, smooth muscle cells and macrophages.^10,11 Macrophages proliferate within lesions¹² and GM-CSF expression frequently colocalizes with proliferating macrophages¹³ and is important for their survival.¹¹ GM-CSF appears to regulate type VIII collagen biosynthesis in atherosclerotic lesions,¹⁴ stimulates macrophages to produce myeloperoxidase¹⁵ and reactive oxygen species,¹¹ increases matrix metalloproteinases,¹⁶ and reduces macrophage apoE secretion.¹⁷ GM-CSF primed mice produce more proinflammatory cytokines when challenged with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-.¹⁸ GM-CSF also possesses potential anti-atherosclerotic properties. Pharmacological doses lower plasma cholesterol levels, reduce liver cholesterol biosynthesis,¹⁹ elevate expression of very-low density lipoprotein (VLDL) receptor,²⁰ and decrease scavenger receptor expression on cultured human monocytes, leading to reductions in cholesterol ester accumulation.²¹ Monocytes stimulated with GM-CSF produce high levels of IL-1 receptor antagonist protein,^22,23 increase expression of peroxisome proliferator-activated receptor- (PPAR-),^24,25 and suppress interferon (INF)- action.²⁶ GM-CSF also induces monocytes to secrete soluble VEGF receptor-1, preventing VEGF-A signaling and angiogenesis.²⁷ Administration of supraphysiological concentrations of GM-CSF reduces atherosclerosis, smooth muscle cell numbers, and collagen content.²⁸ However, despite multiple lines of evidence for a role of GM-CSF in atherosclerosis, the impact and role of physiological levels of endogenous GM-CSF on atherosclerotic lesion formation remain hitherto unknown.

To determine the role of GM-CSF in atherosclerosis, we crossed GM-CSF-deficient mice (GM-CSF^–/–) with apoE^–/– mice. We found that GM-CSF deficiency resulted in larger lesions with increased macrophage accumulation and reduced collagen content. This was accompanied by reduced expression in lesions of PPAR- and ABCA1 and increased expression of proinflammatory TNF-, MCP-1, and VCAM-1. As PPAR- and ABCA1 were also reduced in peritoneal macrophages from GM-CSF-deficient mice, we suggest that reduction of PPAR- and ABCA1 in lesional macrophages is the basis for the larger atherosclerotic lesions in these mice. Our data suggest that GM-CSF has a PPAR--dependent protective role in atherogenesis.

	Methods

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Animals
The generation of GM-CSF^–/– mice on a C57BL6 background used in this study has been described previously.²⁹ These mice exhibit no perturbation of major hematopoietic populations in marrow or blood. apoE^–/– mice on a C57BL6 background were obtained from the Walter and Eliza Hall Institute, Melbourne, Australia. GM-CSF^–/– mice were crossed with apoE^–/– mice and the apoE^+/–.GM-CSF^+/– mice backcrossed to produce apoE^–/–.GM-CSF^–/– mice. Male mice were fed a high-fat diet consisting of 20% butter fat and 0.125% cholesterol from 8 weeks of age for 12 weeks. After pentobarbitone overdose, blood was collected by intracardiac puncture. The aortic sinus was dissected and collected for histological and mRNA analyses. All experiments were approved by AMREP Animal Ethics Committee.

For Materials and Methods used in this article, please see http://atvb.ahajournals.org.

	Results

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GM-CSF Is Upregulated in Aortic Sinus Lesions of ApoE^–/– Mice
GM-CSF was expressed in a temporal manner during development of atherosclerosis in apoE^–/– mice (Figure 1). In nonatherosclerotic aortic sinus, no expression of GM-CSF was observed using immunohistochemistry to detect GM-CSF-positive cells or by reverse-transcription polymerase chain reaction (RT-PCR) analysis of mRNA. GM-CSF-expressing cells became detectable in lesions 4 weeks after mice commenced a high-fat diet, peaking at 8 weeks, and then slowly declining by 50% at 12-weeks (Figure 1C). RT-PCR analysis of mRNA confirmed this time course of expression with GM-CSF transcripts peaking at 8 weeks and then declining (Figure 1D).

View larger version (26K): [in this window] [in a new window]   Figure 1. GM-CSF expression in developing aortic atherosclerotic lesions. Immunohistochemical staining for GM-CSF in developing lesions of the aortic sinus. A, Lack of staining using non-immune IgG control; section counterstained with hematoxylin. B, GM-CSF is represented by the brown stain (indicated with arrows) in sections counter-stained with hematoxylin. C, Quantitative analysis of GM-CSF-positive cells during lesion development after feeding apoE–/– mice a high-fat diet (HFD) for 4, 8, and 12 weeks. Bar graphs represent number of cells/field expressing GM-CSF in developing lesions of the aortic sinus. T0 represents apoE–/– mice not on a HFD. Results are means±SEM of at least 5 mice in each group; +P<0.05. D, Electrophoretic gel showing cDNA fragments specifically detecting GM-CSF mRNA by RT-PCR in mouse aortic atheroma of apoE–/– mice fed a HFD. The expected size of the RT-PCR product of GM-CSF mRNA is 387bp. –RT represents samples subjected to PCR without RT.

Generation of ApoE^–/– Mice Deficient in GM-CSF
To determine the role of GM-CSF in atherogenesis, we crossed GM-CSF^–/– mice with apoE^–/– mice to generate apoE^–/–.GM-CSF^–/– mice. Mice were genotyped with primers specific for wild type and the dysfunctional gene of GM-CSF and apoE (supplemental Figure IA, available online at http://atvb.ahajournals.org). After 12 weeks on a high-fat diet, body weights of apoE^–/–.GM-CSF^–/– mice were 4% higher than apoE^–/– mice (P<0.05; supplemental Figure IB); plasma cholesterol averaged 40 mmol/L and were not significantly different between the 2 groups (P>0.05; supplemental Figure IC).

Atherosclerotic Plaques Are Larger in GM-CSF-Deficient Mice
Cross-sections from lesions in the aortic sinus region were stained with Oil Red-O and lesion area quantified. Plaques from apoE^–/–.GM-CSF^–/– mice were on average 30% larger than plaques from apoE^–/– mice (0.14±0.035 mm² per section and 0.08±0.02 per section, respectively; P<0.05; Figure 2). This increase in lesion size was independent of plasma cholesterol.

View larger version (35K): [in this window] [in a new window]   Figure 2. Atherosclerotic lesions in apoE–/–.GM-CSF–/– mice. Oil red-O staining of the tunica intima of the aortic sinus in apoE–/– mice (A) and apoE–/–.GM-CSF–/– mice (B) fed a high-fat diet. C, Bar graph show the means±SEM from at least 16 mice from each group which were fed a high fat diet. +P<0.05 from apoE–/– mice.

Macrophage Accumulation Is Increased and Collagen Content Decreased in GM-CSF-Deficient Lesions
To evaluate whether the lack of GM-CSF affects inflammatory processes in the lesions, we initially assessed macrophage accumulation. On average, macrophage accumulation in lesions at the aortic sinus of apoE^–/–.GM-CSF^–/– mice was nearly double the accumulation in apoE^–/– mice (Figure 3A; P<0.05). Deletion of GM-CSF did not affect smooth muscle cell numbers in the lesions, which mostly covered the macrophage-rich lesions but were also apparent to a lesser extent within lesions (Figure 3B; P>0.05). As GM-CSF has been reported to affect vascular collagen content,^14,28 we stained sections with picro-sirius red and examined collagen under normal and polarized light. Collagen content in lesions of the apoE^–/–.GM-CSF^–/– mice was 15% lower than in lesions of apoE^–/– mice (P<0.05; Figure 3C). Collagen structure, visually analyzed under polarized light, was different in the apoE^–/–.GM-CSF^–/– lesions with smaller, more disorganized fibrils dominating, indicated by a shift to the violet side of the visible spectrum. The distribution of collagen within lesions appeared to be similar in the 2 groups of mice (Figure 3C).

View larger version (65K): [in this window] [in a new window]   Figure 3. Characteristics of lesions in apoE–/–.GM-CSF–/– mice. A, Photomicrographs together with mean data demonstrating macrophage accumulation (CD68-positive cells, brown stain) in the tunica intima of the aortic sinus in apoE–/– and apoE–/–.GM-CSF–/– mice fed a high-fat diet. B, Photomicrographs with mean data demonstrating smooth muscle cell content (alpha SM actin-positive cells, brown stain) in the tunica intima, measured as alpha smooth muscle actin immunostaining (% tunica intima area). C, Photomicrographs (polarized light) and mean data quantitating collagen content in the tunica intima measured as picro-sirius red staining (% tunica intima area). Bar graphs show means±SEM of at least 13 mice from each group. +P<0.05 from apoE–/– mice.

PPAR- and ABCA1 Expression Is Reduced in GM-CSF-Deficient Lesions and in Peritoneal Macrophages
Because PPAR- is deficient in alveolar macrophages of patients with alveolar proteinosis, an autoimmune disease with high levels of circulating anti-GM-CSF neutralizing antibodies,³⁰ we examined using real-time PCR whether PPAR- was reduced in lesions of apoE^–/–.GM-CSF^–/– mice. We found that PPAR- mRNA expression was reduced by 50% in lesions of apoE^–/– mice that were GM-CSF-deficient compared with apoE^–/– mice (P<0.05, n=5 per group; Figure 4A). Because PPAR- is a potent transcriptional regulator of genes governing lipid metabolism we next examined whether the reduction in PPAR- affected expression of these genes. In lesions of apoE^–/–.GM-CSF^–/– mice, we found that mRNA expression of ABCA1, the ATP-binding cassette protein responsible for cholesterol efflux, was reduced by 65% (P<0.05), the scavenger receptor SR-A tended to be increased and the scavenger receptor CD36 reduced by 55% (P<0.05; Figure 4A).

View larger version (16K): [in this window] [in a new window]   Figure 4. Real-time RT-PCR for mRNA encoding molecules relevant to lipid metabolism and transport. A, mRNA levels encoding PPAR-, SR-A, CD36, and ABCA1 in lesions of apoE–/–.GM-CSF–/– mice relative to apoE–/– mice fed a high-fat diet. Bar graphs represent mean±SEM from 5 mice per group. B, mRNA levels encoding PPAR-, ABCA1, and CD36 in peritoneal macrophages isolated from apoE–/–.GM-CSF–/– compared with apoE–/– mice. Bar graphs represent the means ± SEM of 3 mice per group. C, Plasma initiated cholesterol efflux from thioglycollate-elicited macrophages of apoE–/–.GM-CSF–/– and apoE–/– mice. Bar graphs represent the means ± SEM of at least 3 mice from each group. +P<0.05 from apoE–/– mice.

To confirm that macrophages are responsible for the decrease in PPAR- expression in lesions, we analyzed expression of PPAR-, ABCA1, and CD36 in peritoneal macrophages. PPAR- mRNA levels in peritoneal macrophages were reduced by 50% and expression of ABCA1 was reduced by 70% (P<0.05; Figure 4B); CD36 levels also tended to be reduced. To determine whether the reduction in ABCA1 expression was associated with reduced cholesterol efflux, we compared [³H]-cholesterol efflux from thioglycollate-elicited peritoneal macrophages from apoE^–/–.GM-CSF^–/– and apoE^–/– mice. Cholesterol efflux was reduced by 50% in macrophages from the apoE^–/–.GM-CSF^–/– mice (P<0.05; Figure 4C).

MCP-1, TNF-, and VCAM-1 Expression Is Increased in GM-CSF-Deficient Lesions
Because PPAR- was reduced in lesions of apoE^–/–.GM-CSF^–/–mice and has been reported to suppress inflammatory cytokines,³¹ we next investigated whether expression of inflammatory cytokines in lesions of these mice were increased. Analysis of mRNA from lesions of apoE^–/– and apoE^–/–.GM-CSF^–/– mice indicated increased expression of monocyte chemotactic protein-1 (MCP-1) and TNF-, 2- and 3.5-fold, respectively, in the GM-CSF-deficient mice (P<0.05; Figure 5A), whereas expression of the IL-1 receptor antagonist was reduced by nearly 50% (P<0.05; Figure 5A). VCAM-1 is important in the progression of atheroma and a marker of inflammation.³² Consequently, we also examined its expression in lesions. VCAM-1 expression was also higher in the apoE^–/–.GM-CSF^–/– mice, expressed in 25% of the lesions compared with 12% in lesions of apoE^–/– mice (P<0.05; supplemental Figure II).

View larger version (16K): [in this window] [in a new window]   Figure 5. Real-time PCR analysis of mRNAs encoding inflammatory factors expressed in aortic sinus lesions of apoE–/–.GM-CSF–/– and apoE–/– mice. Relative amounts of mRNA encoding MCP-1, TNF- and IL-1 receptor antagonist (IL-1rn) in the lesions of apoE–/–.GM-CSF–/– and apoE–/– mice were calculated using comparative CT values. Results are means±SEM of 5 mice from each group. +P<0.05 from apoE–/– mice.

Vascularization of Lesions Is Not Affected by GM-CSF Deficiency
GM-CSF has been reported to induce secretion of soluble VEGF receptor-1, thereby preventing VEGF-A signaling and angiogenesis,²⁷ and angiogenesis inhibitors reduce lesion growth in apoE^–/– mice.³³ We examined whether such a mechanism could contribute to the increase in lesion size in the GM-CSF-deficient apoE^–/– mice. Soluble VEGF receptor-1 (sFlt-1) mRNA expression was reduced by 20% and there was a small (10%) increase in expression of VEGF receptor-1 mRNA but the differences were not statistically significant (P>0.05; supplemental Figure III). The number of microvessels in lesions, detected immunohistochemically using anti-CD31 antibodies was also unaffected (P>0.05; supplemental Figure III).

	Discussion

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Accumulated evidence from in vitro studies suggest that GM-CSF is pro-atherogenic^13,31 Here, we found that lesion size and macrophage accumulation is increased in apoE^–/– mice deficient in GM-CSF, suggesting that, in vivo, GM-CSF protects against atherosclerosis.

Our observation of reduced PPAR- expression in atherosclerotic lesions and in peritoneal macrophages of GM-CSF-deficient apoE^–/– mice suggest that the anti-atherogenic action of GM-CSF may be mediated by macrophage PPAR-, which regulates cholesterol metabolism and attenuates inflammation.³⁴ Our data are consistent with the PPAR- deficiency in alveolar macrophages of patients with alveolar proteinosis, an autoimmune disease with high circulating anti-GM-CSF neutralizing antibodies that cause GM-CSF deficiency.³⁰ PPAR- is a nuclear transcription factor that is highly expressed in macrophages and macrophage-derived foam cells in atherosclerotic lesions. PPAR- inhibits macrophage foam cell formation and atherosclerosis³⁵ and its deletion in macrophages increases atherosclerosis in low-density lipoprotein receptor-deficient mice.³⁶ Whereas PPAR- promotes monocyte/macrophage differentiation and uptake of oxidized LDL by enhancing CD36 expression,³⁷ it attenuates SR-A expression³⁸ and induces cholesterol removal from macrophages by promoting ABCA1 expression and function.³⁹ Conversely, disruption of the PPAR- gene lowers expression of ABCA1 in macrophages and reduces cholesterol efflux.⁴⁰ Our finding that ABCA1 expression is also reduced in atherosclerotic lesions and in peritoneal macrophages of GM-CSF and apoE-deficient mice is consistent with GM-CSF being a regulator of PPAR- expression in macrophages during lesion development. The reduced ABCA1 expression suggests impaired reverse cholesterol transport as one mechanism responsible for the larger lesions in the double knockout mice.⁴⁰ Our suggestion is consistent with the report that increased ABCA1 expression in transgenic mice protects against atherosclerosis while its deletion from leukocytes results in significantly larger and more advanced lesions.^41,42 Whereas the significance of CD36 and SR-A for lesion development is currently unclear,⁴³ our findings that CD36 expression is downregulated and SR-A appeared upregulated provides further support for GM-CSF as a regulator of PPAR- expression and function in developing atherosclerotic lesions. Our finding that lesion area in GM-CSF-deficient mice assessed by macrophage accumulation was greater than that assessed by Oil Red-O staining suggests that GM-CSF might also influence the ratio of free-to-esterified cholesterol deposited in lesions. Both nonesterified and esterified cholesterol are deposited in lesions and Oil Red-O only detects the esterified forms.^44,45 Also, oxidized LDL increases free cholesterol accumulation in macrophages.⁴⁶ Further investigations are warranted to determine whether GM-CSF influences the nature of cholesterol that accumulates in lesions. Together, our data suggest that endogenous GM-CSF plays a major role in regulating cholesterol metabolism in macrophages; one mechanism involves promoting PPAR- expression that in turn induces ABCA1 expression.

ABCA1 deletion also increases plasma MCP-1 and TNF- levels, suggesting that it regulates responses to inflammatory stimuli.⁴⁷ Previous studies suggest that GM-CSF contributes to inflammation through monocyte recruitment, increased cell survival, and/or priming macrophages for activation.⁷ For example, GM-CSF enhances LPS-induced and TNF-–induced cytokine production and stimulates IL-1 production by macrophages.¹⁸ In contrast, our studies suggest that GM-CSF can also exert anti-inflammatory effects. We found increases in MCP-1 expression in lesions of apoE^–/– mice deficient in GM-CSF, consistent with the report that elevated MCP-1 accelerates atherosclerosis, likely by promoting macrophage accumulation.⁴⁸ Because ABCA1 deficiency in macrophages enhances their responses to MCP-1,⁴⁸ it is possible that reduced ABCA1 in macrophages contributed to macrophage accumulation and development of larger lesions in the GM-CSF-deficient mice. Moreover, we found that TNF- expression was elevated in these larger lesions. Because TNF- is increased in mice deficient in ABCA1⁴² and ABCA1 is reduced in lesions of apoE^–/– mice deficient in GM-CSF, it is likely that the increase in TNF- is also mediated by an ABCA1-dependent mechanism. Further, we found expression of IL-1 receptor antagonist was reduced, consistent with the report that monocytes stimulated with GM-CSF elevated levels of IL-1 receptor antagonist.²³ Our finding of enhanced VCAM-1 expression in atherosclerotic lesions of GM-CSF-deficient mice provides further support for our suggestion of an anti-inflammatory role for GM-CSF in developing atherosclerotic lesions.

Whereas GM-CSF deficiency did not affect smooth muscle cell content in lesions, it reduced collagen content in lesions, consistent with a stimulatory effect of GM-CSF on collagen production in blood vessels^14,49 and contrasting with the effects of administration of supraphysiological amounts of GM-CSF, that resulted in reductions in both smooth muscle cell numbers and collagen content.²⁸ GM-CSF has been reported to increase collagen expression in cultured airway smooth muscle cells by inducing transforming growth factor-ß receptors.⁵⁰ It is possible that GM-CSF acts directly on vascular smooth muscle cells within lesions via a similar mechanism to elevate collagen expression in lesions. Thus, endogenous GM-CSF may also regulate lesion development by promoting more fibrotic and stable atherosclerotic lesions. Although GM-CSF has been reported to inhibit angiogenesis by stimulating expression of soluble VEGF receptor-1 in monocytes,²⁷ vascularity was unaffected in developing lesions in double knockout mice.

In conclusion, we have demonstrated that GM-CSF deficiency led to an increase in size of atherosclerotic lesions in diet-induced hyperlipidemic apoE^–/– mice. The increase in lesion size and macrophage accumulation in GM-CSF-deficient mice appears due to reductions in PPAR- and ABCA1 expression in macrophages. Our results suggest that endogenous GM-CSF modulates lesion development during hyperlipidemia resulting in smaller more stable lesions.

	Acknowledgments

 
Source of Funding

This work was supported by an NHMRC program grant (ID225108) held by Alex Bobik.

Disclosures

None.

	Footnotes

 
B.-H.T. and A.B. contributed equally to this work.

Original received February 9, 2006; final version accepted July 9, 2006.

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