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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:571-577
Published online before print December 28, 2006, doi: 10.1161/01.ATV.0000256470.23842.94
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:571.)
© 2007 American Heart Association, Inc.

Vascular Biology

Genomics of Foam Cells and Nonfoamy Macrophages From Rabbits Identifies Arginase-I as a Differential Regulator of Nitric Oxide Production

Anita C. Thomas; Graciela B. Sala-Newby; Yasmin Ismail; Jason L. Johnson; Gerard Pasterkamp; Andrew C. Newby

From Bristol Heart Institute (A.C.T., G.B.S.-N., Y.I., J.L.J., A.C.N.), University of Bristol, Bristol, UK; University Medical Center Utrecht (G.P.), Experimental Cardiology Laboratory, Utrecht, The Netherlands. Current address for A.C.T.: Centre for Research in Vascular Biology, University of Queensland, Brisbane, Australia.

Correspondence to Graciela B. Sala-Newby, Bristol Heart Institute, University of Bristol, 7th Floor, Bristol Royal Infirmary, Bristol, BS2 8HW, United Kingdom. E-mail g.newby{at}bristol.ac.uk


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Objective— Conversion of macrophages to foam cells is a critical step in the initiation and progression of atherosclerosis. We sought to identify genes differentially regulated in foam cells, since these are likely to include new targets for intervention.

Methods and Results— We used suppression subtraction hybridization to compare foam cells and nonfoamy macrophages isolated from subcutaneous granulomas of rabbits fed a cholesterol-rich or normal chow diet and confirmed upregulation of 3 genes, including matrix metalloproteinase-12 (mRNA 2.0-fold, P<0.005; protein 3.9-fold, P<0.03). Arginase-I mRNA showed the biggest decrease among 11 downregulated genes in foam cells (2.7-fold, P<0.001) and was accompanied by significantly reduced arginase enzymatic activity (60-fold, P<0.01). Arginase-I competes for substrate L-arginine with nitric oxide synthase and consequently nitric oxide production was significantly increased (3-fold, P<0.02) in foam cells compared with nonfoamy macrophages despite no difference in nitric oxide synthase isoenzyme expression. We validated upregulation of matrix metalloproteinase-12 and downregulation of arginase-1 in foam cells of rabbit and human atherosclerotic plaques.

Conclusions— Our study identified several differentially expressed genes in foam cells and nonfoamy macrophages derived from live rabbits. The altered pattern of gene expression in foam cells is likely to influence atherosclerosis formation and stability.

Suppression subtraction hybridization showed upregulation of 3 and downregulation of 11 genes in rabbit foam cells compared with nonfoamy macrophages. FCMs had more MMP-12 but less arginase-I activity, leading to more NO production, which probably influences atherosclerosis.


Key Words: arginase-I • atherosclerosis • foam cells • nitric oxide • suppression subtraction hybridization


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
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Atherosclerosis is initiated by deposition into the artery wall of cholesterol-rich lipoproteins, which become oxidized and taken up into macrophages. The resulting foam cells are believed to play several key roles in orchestrating atherogenesis.1,2 For example, secretion of cytokines and growth factors from foam cells most likely initiates fibrous cap formation. In the late stages, overproduction of inflammatory mediators, reactive oxygen species including nitric oxide (NO) and extracellular proteases is believed to result in cell death and net destruction of extracellular matrix. Together these lead to lipid core formation and thinning of the fibrous cap, both of which favor plaque instability and myocardial infarction.3,4

Genomic methods have been applied to atherosclerotic and normal tissues with the hope of identifying disease-related genes to further elucidate pathogenetic mechanisms and define new targets for intervention.5,6 A major obstacle for characterizing atherosclerotic tissue is however its heterogeneous cellular composition of endothelial cells, smooth muscle cells, macrophages, T-lymphocytes, fibroblasts, mast cells, neutrophils, erythrocytes, and a variety of secreted proteins.1 An alternative has been to study vascular cells exposed to inflammatory mediators or oxidized lipoproteins in vitro.7–9 However, these interventions inevitably recapitulate only part of the in vivo process of foam cell formation. In this study we took advantage of established methods to simply isolate abundant pure populations of foam cells and nonfoamy macrophages from subcutaneous sponges implanted into live rabbits fed cholesterol-rich or normal chow diets, respectively.10,11 The foam cells so generated are very similar to those in atherosclerotic plaques.11 We compared expression patterns using suppression subtraction hybridization (SSH), which is suitable for rabbits and is able to detect low-abundance transcripts. Differential expression of genes was confirmed by semi-quantitative polymerase chain reaction (PCR) before selecting candidates for protein expression and functional investigations. Finally, we validated differential expression of selected genes in foam cells of atherosclerotic plaques.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
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Please see details in the online supplement available at http://atvb.ahajournals.org.

New Zealand White rabbits (Harlan, UK) fed a 1% cholesterol-supplemented or normal chow diet had sterile sponges placed under the dorsal skin to generate foam cell macrophages (FCMs) or nonfoamy macrophages (NFMs), respectively, as described previously.10 Endarterectomy specimens were made available from the AtheroExpress biobank.12 Human blood mononuclear cells differentiated with 20 ng/mL human macrophage colony-stimulating factor at 37°C for 8 days were incubated with 100 µg/mL fully oxidized low-density lipoprotein (LDL) to generate FCMs.

SSH was performed on equal amounts of total RNA pooled from 2 and 3 preparations of FCMs and NFMs, respectively, using the PCR-Select subtraction kit (BD Biosciences) according to the manufacturer’s instructions. Clones showing differential expression were sequenced and homology searches were performed using Mega Blast against all GeneBank DNA sequences. Semi-quantitative reverse-transcription PCR was performed for various numbers of cycles to avoid saturation. Products separated on agarose gels were analyzed by densitometry and normalized to 18S rRNA.

Hydrolysis of arginine to urea was measured in cell lysates and normalized against cell number or protein levels (bicinchoninic acid protein assay, Pierce). NO was measured as nitrite by a modified Griess assay.

Immunocytochemistry was conducted on 5 µm sections from formaldehyde-fixed paraffin-embedded tissues using mouse monoclonal RAM-11 (anti-rabbit alveolar macrophage; Dako), monoclonal anti-human CD68 antigen (Dako), or anti-human arginase-I (cat 612621; BD Biosciences).

Data were compared using the unpaired Student t test or the Mann-Whitney test, with or without logarithmic transformation as indicated.


*    Results
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Cell Yield, Purity, and Viability
Sponges yielded 18±3x106 (range, 1.5 to 60x106; n=24) FCMs and 4±1x106 (range, 0.8 to 12x106; n=13) NFMs per rabbit, similar to previously reported values.10,11 FCMs were >98% Oil red O-positive, confirming cholesterol loading, whereas NFMs did not stain (Figure 1A, 1B). FCMs and NFMs consisted of >98% macrophages by RAM11 positivity (Figure 1C, 1D). Day 3 cultures of FCMs and NFMs were 99% viable by exclusion of Trypan blue (results not shown).


Figure 1
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  		Figure 1. Characteristics of rabbit FCMs and NFMs. A, Oil red O staining of FCM. B, Oil red O staining of NFMs. C, RAM11 staining (brown) of FCMs. D, RAM11 staining of NFMs. E, Nonimmune control for FCMs. F, Nonimmune control for NFMs. Counterstain is hematoxylin (blue). Original magnification 400x.

SSH
The libraries generated from the forward (upregulated in FCMs) and reverse (upregulated in NFMs) subtraction contained 1300 and 1344 clones, respectively. Initial radioactive screening identified 88 and 147 clones that were potentially overexpressed in FCMs and NFMs, respectively, of which 47 and 114 were found to be unique.

Upregulated Genes
At least 12 different FCMs and NFMs isolates were used to produce cDNA for semi-quantitative PCR. Because GAPDH was found to be upregulated in NFMs preparations (1.8 fold, n=8/group; P=0.023), 18S rRNA levels were used to normalize our results (P=0.94; n=8/group). Six cDNA preparations from FCMs and NFMs were compared in triplicate under linear conditions. Of 27 candidate genes tested from the forward subtraction, we confirmed upregulation of 3 (Table 1), a rate of 89% false-positives. To validate the mRNA data we used Western blotting and demonstrated an increase in MMP-12 protein secretion from FCMs versus NFMs (14.8±2.7 versus 3.8±2.6 U, n=4; P=0.03).


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  		TABLE 1. Genes Upregulated in Foam Cell Macrophages

Downregulated Genes
Of 23 genes we tested from the reverse subtraction, we found 11 significantly downregulated by semi-quantitative PCR (Table 2), a rate of 52% false-positives. The most significant decrease observed was the 2.7-fold lower mRNA for arginase-I in FCMs compared with NFMs (1.5±0.2 versus 3.9±0.5, 30 cycles, n=6/group; P=0.001; Table 2). By contrast, no change in arginase-II expression was detected (2.6±0.7 versus 3.0±0.6, 36 cycles, n=6/group; P=0.74). Arginase converts L-arginine to urea and ornithine hence we measured urea production from arginine to investigate whether differential arginase-I mRNA expression led to a difference in arginase activity in FCMs and NFMs homogenates. There was no urea production without the addition of arginine (not shown). Arginase activity in NFMs preparations was highly variable (Figure 2A, 2B). However, FCMs had significantly less arginase activity based either on protein content (10 versus 610 nmol · min–1 · mg protein–1; P=0.0043) or cell number (0.12 versus 7.3 nmol · min–1 · 106cells–1, n=6/group; P=0.0087).


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  		TABLE 2. Genes Upregulated in Nonfoamy Macrophages


Figure 2
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  		Figure 2. Arginase activity and NO production from rabbit FCMs and NFMs. Homogenate arginase activity measured as (A) urea production nmol · min–1 · mg of protein–1 or (B) nmol · min–1 · 106 cells–1. Whole cell nitrite production (C) nmol · 48 hours–1 · mg of protein–1 or (D) nmol · 48 hours–1 · 106 cells–1. Results were compared using the Mann-Whitney test.

Because arginase-I and nitric oxide synthase (NOS) compete for L-arginine as substrate, we hypothesized that NO synthesis might be increased in FCMs compared with NFMs. Indeed, NO production measured as nitrite accumulation was significantly greater in the medium conditioned by FCMs than NFMs (12.6 versus 4.9 nmol · 48 hours–1 · mg protein–1; P=0.015 and 0.54; versus 0.13 nmol · 48 hours–1 0.106 cells–1, P=0.001; n=14 and 10, respectively). Consistent with the variable arginase levels in NFMs some preparations had very low levels of nitrite production (Figure 2C, 2D). We considered whether differential expression of NOS isoenzymes might provide an alternative explanation for these data. There was, however, no difference in nNOS (1.42±0.18 versus 1.30±0.20; P=0.68) or iNOS (1.45±0.35 versus 1.83±0.22; P=0.38) mRNA levels between FCMs and NFMs, whereas no mRNA for eNOS was found. We concluded that decreased arginase-I activity in FCMs led to an increase in arginine availability to NOS isoforms and hence increase NO production.

Validation of Differential Gene Expression in Atherosclerotic Plaques and Oxidized LDL-Treated Human Macrophages
Bulk measures of mRNA and protein could not be relied on to validate differential expression in atherosclerotic plaque macrophages because plaques also contain other cell types. Therefore, we had no choice but to rely on immunocytochemistry. We chose to study MMP-12 and arginase-I as an example of an upregulated and a downregulated gene because antibodies were available for both proteins. MMP-12 was undetectable in normal rabbit arteries (not shown). Weak staining for MMP-12 was found in foam cells of fatty streaks (Figure 3A to 3D). MMP-12 staining was also weak in macrophages in the superficial layers of advanced rabbit plaques (supplemental Figure I, available online at http://atvb.ahajournals.org) but was abundant and tightly co-localized with FCMs deep within the plaques (Figure 4E to 4H). This pattern replicates the distribution of MMP-12 previously reported in human atherosclerotic plaques.13,14 Arginase-I was found in foam cell macrophages in fatty streaks (Figure 4A, 4B) but was also found in nonmacrophages, presumably smooth muscle cells (arrows in Figure 4A). In advanced rabbit atherosclerotic plaques, arginase-I was also present in the superficial nonfoamy macrophages (arrows in Figure 4C) and adventitial nonfoamy macrophages (Figure 4E). It was also expressed by foam cells and nonmacrophage cells underlying the fibrous cap of advanced plaques (above the dotted line in Figure 4C). However, arginase-1 was downregulated in subjacent FCMs deeper within the plaques (below the dotted line in Figure 4C and 4D), despite the strong RAM11 positivity of both superficial and deep foam cells (Figure 4D). In human carotid atherosclerotic plaques, arginase-I was also widely distributed in the superficial cell layers (eg, above the dotted line in Figure 4G), including in CD68-positive macrophages (Figure 4H). However, arginase-I was absent from macrophages in subjacent layers (eg, below the dotted line in Figure 4G), despite the abundance of CD68 staining (Figure 4H). In sections from other plaques arginase-I was consistently absent from macrophages that were close to or within a lipid core (not shown).


Figure 3
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  		Figure 3. MMP-12 localization in rabbit atherosclerotic plaques. A, Peroxidase stain for macrophages with RAM11 in a 4-week rabbit fatty streak. The box shows the area chosen for dual labeling in (B), (C), and (D). B, RAM11 immunofluorescent staining in red. C, MMP-12 immunofluorescent staining in green. D, Images (B) and (C) superimposed where co-localization is yellow. E, Peroxidase stain for macrophages with RAM11 an 8-week advanced atherosclerotic plaque. The box shows the area chosen for dual labeling in (F to H). F, RAM11 immunofluorescent staining in red. G, MMP-12 immunofluorescent staining in green. H, Images (F) and (G) superimposed where co-localization is yellow. Scale bar=100 µm. A representative of n=4 each.


Figure 4
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  		Figure 4. Arginase-I and macrophage localization in rabbit and human atherosclerotic plaques. Serial sections from a 4-week fatty streak lesion stained with Arg-I (A) or RAM11 (B). Macrophages and nonmacrophage cells (arrows) stain for ARG-I. In sections from an 8-week advanced plaque stronger immunostaining for ARG-I (C) occurs in nonfoamy macrophages near the lumen (arrows) and in the foam cell macrophages above the dotted line (C and D). Strong ARG-I staining also occurs in nonfoamy adventitial macrophages (arrows in E and F). In sections from a human carotid atherosclerotic plaque, there is strong staining for ARG-I (G) in CD68-positive macrophages (H) and nonmacrophages in the superficial layers but comparatively weak immunostaining for ARG-I in the macrophages surrounding the lipid core of a human plaque. A representative of n=4. Scale bar = 100 µm.

Neither MMP-12 nor arginase-1 mRNA levels were significantly changed when human monocyte-derived macrophages were treated with oxidized LDL for 5 days in vitro although other positive control genes including MMP-1 were upregulated (results not shown).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Main Findings
Ours is the first genomic study we are aware of to compare foamy and nonfoamy macrophages produced in vivo. SSH identified 3 novel upregulated and 11 downregulated genes in foam cells. Using MMP-12 as an example, we confirmed that increase mRNA levels translated into increased protein secretion for this gene. Using arginase-I (the most downregulated gene) as a second example, we validated that the difference in gene expression translated into a difference in enzymatic activity. Moreover, we showed that this had functional consequences for NO production, which is believed to play an important role in atherogenesis. Finally, we validated that MMP-12 was upregulated and arginase-I downregulated in a specific population of FCMs in advanced atherosclerotic plaques.

Advantages and Disadvantages of Our Approach Compared With Previous Studies
Because foam cell formation is believed to be a key event in atherosclerosis, several previous approaches have been applied directly or indirectly to define the foam cell transcriptome. Genomic studies comparing atherosclerotic plaques and normal tissues found many marker genes for activated macrophages.15 However, such studies probably reflect the different proportion of cells present rather than changes within any particular cell type.9,16 Laser microdissection allowed the isolation of plaque macrophages for genomic studies and differentially expressed genes have been identified.9,15,17 However, dissected samples are too small for metabolic studies to investigate the functional significance of these changes.5,6 Therefore, we were keen to exemplify (using arginase-I) the possibility that such metabolic studies can be performed using our approach. An alternative has been to simulate macrophage activation and the formation of foam cells in cultured human monocytes/macrophages or cell lines. The limitation of these in vitro studies is that they do not necessarily replicate the complete process of foam cell formation in vivo.

Comparison of Our Findings With Previous Literature
In our SSH screen, we found that 161 genes appeared differentially regulated in FCMs and NFMs derived from rabbits given a cholesterol-rich or normal diet, respectively. We further tested 50 of these using reverse-transcription PCR and confirmed differential expression for 14 genes, a proportion of false-positives that is comparable to other studies.16,18,19 It is difficult to compare directly results from SSH with microarrays because SSH is less comprehensive and genes belonging to families can be cross-subtracted and therefore missed. However, SSH can be used in species such as rabbits where the genome is not yet fully defined. SSH also potentially identifies low-abundance genes that might be missed in arrays. With these limitations we found changes in expression of the same categories of genes as microarray studies.

Among the proteolytic enzymes, we found that MMP-12 was upregulated in FCMs compared with NFMs. Interestingly, MMP-12 knockout decreases atherosclerosis and plaque vulnerability in mice,20 whereas overexpression of MMP-12 promotes plaque progression in transgenic rabbits.14 Hence, upregulation of MMP-12 during foam cell formation appears to play an important pathogenetic role. We found that loading human monocyte derived macrophages with oxidized LDL did not change MMP-12 expression. From previous studies, differentiation of human monocytes to macrophages or THP-1 cells with PMA significantly increased expression of many genes including MMP-9 and MMP-14. However, addition of mildly oxidized LDL did not produce any further changes in gene expression.21 Stimulating PMA differentiated THP-1 cells with fully oxidized LDL increased expression of several extracellular proteases and decreased that of others, but MMP-12 was not included in either category.8 Liang et al14 also reported that oxidized LDL failed to stimulate MMP-12 production from U937-derived macrophages. This implies that MMP-12 upregulation occurs in macrophages that have undergone lipid loading plus additional phenotypic changes.

In addition, we found that a proteasome subunit, a peptidase, and cathepsin K were downregulated in FCMs compared with NFMs. An association between foam cell formation and lack of cathepsin K in mice has been recently reported.22 The upregulation of LOX-1 during foam cell formation is a novel finding but is supported by previous studies showing that LOX-1 is present in macrophages in advanced human atherosclerotic lesions.23 LOX-1 is upregulated in macrophages by tumor necrosis factor-{alpha},24 advanced glycation products,25 high glucose26 and histamine,27 and downregulated by lovastatin.28 We are presently developing reagents to pursue the functional consequences of LOX-1 upregulation in foam cells. The upregulation of an inhibitor of AKT suggests that FCMs might be more susceptible to apoptosis than NFMs. However, we observed similar high levels of viability and low levels of cleaved caspase-3 staining in FCMs and NFMs (results not shown). The downregulation of adhesion molecules and cytoskeletal proteins (fibronectin, thrombospondin and nesprin) might affect motility and adhesion. Decreased acyl-coenzyme A synthetase expression in FCMs might point toward lower fatty acid metabolism in FCMs. Further studies are needed to address these possibilities.

The most statistically significant change we observed was the average 2.7-fold reduction in arginase-I mRNA in FCMs. Because the correlation between mRNA and protein is often not linear,6 it was important to validate our SSH results with measurements of enzymatic activity, which showed, on average, much lower arginase-I activity in FCMs. Cytosolic arginase-I occurs in hepatocytes, where it is an integral part of the urea cycle. It is also induced in a number of vascular cells, including endothelial cells, smooth muscle cells, and macrophages by Th2 cytokines including IL-4, IL-10, and IL-13, and granulocyte macrophage colony-stimulating factor.29–31 By contrast, arginase-I is downregulated in macrophages by the Th1 cytokine, interferon-{gamma}.31 Neither our study nor a previous genomic analysis found downregulation of arginase-I directly from loading of macrophages with oxidized LDL.8 This complex pattern of regulation may explain the striking biological variability in the arginase activity in NFMs derived from different rabbits (Figure 2A, 2B). Mitochondrial arginase-II is found constitutively at low levels in the mitochondria of most cells,32 consistent with our reverse-transcription PCR data in FCMs and NFMs. Arginase converts arginine to ornithine and urea and competes directly for substrate with NOS. The Km for arginase is 2 to 20 mmol/L compared with 2 to 20 µmol/L for the NOS enzymes, but the Vmax of arginase at physiological pH is generally 1000 times that of the NOS enzymes, which therefore have a similar rate of arginine consumption32; this is the so-called arginine paradox. Indeed, arginase activity in our experiments exceeded the rate of nitrite production by {approx}3000 fold. Consistent with the ability of arginase-I to regulate NO production, we found that FCMs produced 3-fold more nitrite than NFMs (Figure 2C, 2D), even though nNOS and iNOS expression was the same and endothelial NOS was undetectable.

In general, further in vivo experiments, most likely in genetically modified mice, would be needed to investigate the pathological role of differentially expressed genes identified in a genomic screen. However, in the case of macrophage arginase-I, an experiment of nature has already identified a correlation between low levels of the enzyme and atherogenesis. Teupser et al19 recently reported that peritoneal macrophages from an atherosclerosis-prone strain of rabbits had lower levels of arginase-I expression. This implies that lower arginase-I levels favor atherosclerosis. Our new data show that foam cell formation further lowers arginase-I levels, and it would be interesting to compare arginase-I levels in the foam cells of the atherosclerosis prone and wild-type rabbit strains. Whether arginase-I is protective against atherosclerosis simply by reducing NO production is an unanswered question. A wealth of evidence suggests that endothelial NO production (from endothelial NOS) is protective against atherosclerosis by, for example, causing vasodilatation, reducing LDL oxidation, and reducing monocyte recruitment to lesions.33 However, macrophage NO production (most actively from iNOS) is probably harmful,34,35 because iNOS knockout attenuates atherosclerosis in apolipoprotein E knockout mice.36 High levels of NO and superoxide within the lesions creates conditions favorable for formation of peroxynitrite, which may cause injury as a consequence of protein nitration.37 High levels of NO from macrophages also cause apoptosis of smooth muscle cells,34 which could promote plaque instability. It is reasonable to conclude that downregulation of arginase-I and the consequent upregulation of NO production in foam cells most likely enhance their pathological potential.

Validation of Expression Changes in Atherosclerotic Plaques
Previous studies showed a consistent pattern of MMP-1 and MMP-3 expression between granuloma-derived and atherosclerotic plaque foam cells.38 To further validate our approach, we studied MMP-12 and arginase-I as examples of upregulated and downregulated genes. We used immunocytochemistry to overcome the problem of expression from other vascular cells. We found that MMP-12 was not greatly upregulated, nor was arginase-I apparently downregulated in foam cells of early fatty streaks formed after 4 weeks of cholesterol feeding. However, both MMP-12 upregulation and arginase-I downregulation did occur in the deep layers of advanced lesions formed after 8 weeks of cholesterol feeding. Consistent with our data, MMP-12 upregulation was previously reported in foam cells of advanced rabbit atherosclerotic plaques formed after 16 weeks of cholesterol feeding.39 In human plaques, MMP-12 upregulation was reported to be confined to foam cell macrophages surrounding the lipid core of human carotid atherosclerotic plaques.13 We found a similar distribution in human carotid plaques for foam cells with arginase-I downregulation. One possibility we considered is that this gene expression pattern might be selective for foam cells undergoing apoptosis. This is unlikely, however, because, as mentioned, apoptosis was low in both FCMs and NFMs. In addition, a previous study40 demonstrated very low levels of apoptosis in atherosclerotic plaques of rabbits treated according to our protocol. Our immunolocalization studies therefore provide addition support for the conclusion that MMP-12 upregulation and arginase-I downregulation require lipid loading and additional phenotypic changes.


*    Acknowledgments
 
We thank Dr Ray Bush for expert assistance.

Sources of Funding

The work was supported by grants from the British Heart Foundation and the European Vascular Genomics Network. A.C.T. is the recipient of an Australian National Health and Medical Research Council Biomedical (C.J. Martin) Overseas Fellowship (ID #252926).

Disclosures

None.


*    Footnotes
 
A.C.T. and G.B.S.-N. contributed equally to this study.

Original received June 2, 2006; final version accepted December 7, 2006.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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