Published online before print March 8, 2007, doi: 10.1161/01.RES.0000262654.84810.6c
© 2007 American Heart Association, Inc.
Cellular Biology |
Intimal Smooth Muscle Cells of Porcine and Human Coronary Artery Express S100A4, a Marker of the Rhomboid Phenotype In Vitro
From the Department of Pathology and Immunology (A.C.B., M.B., A.G., C.C., G.G., M.-L.B.-P.), University of Geneva, Switzerland; Department of Surgical Pathology (H.H.), Hyogo College of Medicine, Japan; Division of Cardiology (E.C.) and Central Clinical Chemistry Laboratory (J.-C.S.), University Hospital of Geneva, Switzerland.
Correspondence to Dr Marie-Luce Bochaton-Piallat, University of Geneva-CMU, Department of Pathology and Immunology, Rue Michel Servet -1, 1211 Geneva 4, Switzerland E-mail Marie-Luce.Piallat{at}medecine.unige.ch
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Abstract |
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We reported that smooth muscle cell (SMC) populations isolated from normal porcine coronary artery media exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). R-SMCs are recovered in higher proportion from stent-induced intimal thickening compared with media suggesting that they participate in intimal thickening formation. Our aim was to identify a marker of R-SMCs in vitro and to explore its possible expression in vivo. S- and R-SMC protein extracts were compared by means of 2-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry. S100A4 was found to be predominantly expressed in R-SMC extracts. Using a monoclonal S100A4 antibody we confirmed that S100A4 is highly expressed by R-SMCs and hardly detectable in S-SMCs. S100A4 was colocalized with
-smooth muscle actin in stress fibers of several quiescent cells and upregulated during migration. PDGF-BB, FGF-2 or coculture with endothelial cells, which modulate S-SMCs to a R-phenotype, increased S100A4 expression in both S- and R-SMCs. Silencing of S100A4 mRNA in R-SMCs decreased cell proliferation, suggesting a functional role for this protein. In vivo S100A4 was absent in normal porcine coronary artery media, but highly expressed by SMCs of stent-induced intimal thickening. In humans, S100A4 was barely detectable in coronary artery media and markedly expressed in SMCs of atheromatous and restenotic coronary artery lesions. Our results indicate that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs during intimal thickening development. It is also a marker of a large population of human atheromatous and restenotic SMCs. Clarifying S100A4 function might be useful to understand the evolution of atherosclerotic and restenotic processes.
Key Words: 2D-PAGE • stent • endothelial cells • mts1 • -smooth muscle actin • smoothelin
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concept of smooth muscle cell (SMC) phenotypic heterogeneity has been validated in several species including man (for review see1). We have recently extended this notion to the porcine coronary artery (CA) and isolated from the normal media 2 distinct SMC populations: spindle-shaped (S) with the classical "hills-and-valleys" growth pattern and rhomboid (R), which grows as a monolayer.2 R-SMCs display enhanced proliferative, migratory and proteolytic activities as well as poor level of differentiation compared with S-SMCs. R-SMCs are recovered in higher proportion when SMCs are cultured from the intimal thickening (IT) induced after experimental stent implantation compared with the normal media, indicating that they are crucial for arterial repair, and could represent an atheroma-prone phenotype.
Our aim was to further characterize the phenotypic features of S- and R-SMCs, to identify them in vivo and possibly to verify their presence in atheromatous plaque and restenotic lesions. We have analyzed protein extracts by means of 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by identification of differentially expressed proteins using tandem mass spectrometry (MS/MS), according to previous work using rat SMCs that however did not yield markers applicable to the human situation.3,4 This approach has allowed the identification of a new R-SMC marker, the protein S100A4 that belongs to a large family of low molecular weight calcium-binding proteins, of which 21 members have been described so far.5,6 Moreover we show that S100A4 downregulation in R-SMCs is associated with decreased cell proliferation suggesting a functional role of this protein.
Rabinovitch and coworkers7 have shown that S100A4 is expressed in intimal SMCs accumulating during human plexogenic arteriopathy induced by pulmonary hypertension; in addition, S100A4-overexpressing mice display features of this disease.7 Here, we show that S100A4 accumulates in porcine CA intimal SMCs after experimental stent implantation and is neo-expressed in SMCs of human IT, atheromatous plaque and restenotic lesions, suggesting that it represents a hitherto unavailable marker of intimal SMCs implicated in atherosclerosis and restenosis.
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Materials and Methods |
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Animal and Human Specimens
Animal procedures were performed according to federal veterinary guidelines and approved by the Ethical Committee of the Geneva Medical Faculty. Three month-old domestic crossbred pigs (Sus scrofa) were used. IT was induced by direct self-expanding stent implantation (Wallstent, Schneider, Bulach, Switzerland) in the left anterior descending and circumflex CA.8 Injured CA were collected 10 (n=4) and 30 (n=4) days after stent implantation. Nonstented left anterior descending, circumflex as well as right CA served as controls (n=4). Tissue specimens were fixed with 10% neutral buffered formalin and embedded in paraffin or snap-frozen in OTC resin (Miles Scientific, Naperville, Ill).
Human CA slides were selected by 1 of us (H. Hao) from autopsy material of the National Cardiovascular Center, Osaka, Japan. Local research ethic was followed for collection of human material. Specimens (n=29) are described in supplemental Table I in the online data supplement available at http://circres.ahajournals.org. All tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin.
Cell Culture and Treatment
CA of 8 month-old pigs were obtained from a nearby slaughterhouse. SMCs with different phenotypes were isolated from the media using enzymatic digestion (S-SMCs) or tissue explantation (R-SMCs).2 Endothelial cells (ECs) were isolated from CA as previously described.2
Cultured SMCs (n=5 for each phenotype) were treated with heparin, FGF-2, TGF-ß2, or PDGF-BB. SMCs were also plated in serum-free medium (SFM) or cocultured with confluent ECs. SMCs were harvested after 7 days for Western blotting. Migration assay was performed on confluent SMC cultures (n=5 for each phenotype). Experiments were repeated at least 3 times. Detailed experiments are described in the online data supplement available at http://circres.ahajournals.org.
2D-PAGE and Protein Identification
Cell extracts derived from 3 distinct preparations of each SMC phenotype were obtained from 6 different pigs. The 2D-PAGE assays were performed as described in the online data supplement available at http://circres.ahajournals.org.
Antibodies
A monoclonal antibody against S100A4 was produced using a peptide corresponding to the C-terminal 16 amino acids of human S100A4 (NH2-CNEFFEGFPDKQPRKK-COOH, Neosystem, Strasbourg, France) conjugated to keyhole limpet hemocyanin (Pierce Biotechnology, Rockford, Ill). Six 6 week-old Balb/c mice were immunized according to repetitive immunization murine multiple sites strategy.9 Lymphocytes were fused with NSO myeloma cells. Screening was performed by ELISA, Western blotting and immunofluorescence staining. Hybridoma cells secreting S100A4 antibody were subjected to 2 successive limited dilutions. The subclass of S100A4 antibody was determined using a panel of secondary antibodies recognizing different Ig subclasses. The following other primary antibodies were used: 1) a rabbit polyclonal IgG specific for S100A4 (Dako, Copenhagen, Denmark); 2) a mouse monoclonal IgG2a specific for -smooth muscle actin (-SMA, clone 1A4),10; 3) a mouse monoclonal IgG1 specific for smoothelin (clone R4A),11;4) a mouse monoclonal IgG1 specific for CD172A, a marker of porcine macrophages (clone DH59B, VMRB, Pullman, Wash); and 5) a mouse monoclonal IgG1 recognizing all actin isoforms (clone C4, Chemicon, Temecula, Calif).
Protein Extraction, Electrophoresis, and Western Blotting
Cultured SMCs were processed for SDS-PAGE as described in the online data supplement. To further test the specificity of the monoclonal S100A4 antibody, 2D-gels were electro-transferred on PVDF membranes (0.45 µm, Immobilon-P, Millipore Corporation, Bedford, Mass), immunoblotted with the antibody, and revealed as a Western blot.
Immunofluorescence Staining and Immunohistochemistry
Immunofluorescence staining was performed on cryosections of porcine CA or on cultured cells and immunohistochemical staining was performed on paraffin sections of porcine CA and human specimens as described in the on-line data supplement. To identify replicating cells, R-SMCs were incubated with bromodeoxyuridine (BrdU) as described in the online data supplement.
RNA Extraction, Reverse-Transcription and Real-Time Quantitative PCR
Total cellular RNA was prepared from confluent cultured cell dishes (n=7 for each phenotype) with TRIzol reagent (Invitrogen, Basel, Switzerland) and processed for reverse transcription and real-time SYBR Green fluorescent PCR as described in the on-line data supplement. Specific primers for porcine S100A4, ß-interferon (ß-IFN) and GAPDH mRNA were designed as follows: 5'-GGCCCTCGATGTGATGGTGT-3' (reverse) and 5'-GCGATGCAGGACAGGAAGAC-3' (forward) covering nucleotides 63 to 291 of S100A4, 5'-TGAACTTCGAGGTCCCTGAG-3' (reverse) and 5'-CATTCCAGCCAGTGCTAGAG-3' (forward) covering nucleotides 170 to 304 of IFN-ß and 5'-TGGAGTCCACTGGTGTCTTC-3' (reverse) and 5'-TCTCATGGTTCACGCCCATC-3' (forward) covering nucleotides 241 to 370 of GAPDH.
Small Interfering RNA Transfection
Two S100A4-targeting small interfering RNAs (siRNAs) were designed according to Reynolds et al12 and their specificity was assessed by BLAST searches. SiRNAs (21 bp) were homologous to the coding sequence of pig S100A4 from nucleotide position 186 to 206 (P186) and 328 to 348 (P328). A nonsilencing siRNA (scramble) was used as a negative control (10 nM, Qiagen, Hombrechtikon, Switzerland). Transfection of siRNAs (10 nM) was performed on trypsinized R-SMCs using Lipofectamine 2000 (2 µL/mL, Invitrogen) in OptiMEM (Gibco, Invitrogen). R-SMCs were then plated at a density of 13 cells/mm2 in 12-well plates on 15 mm-diameter glass coverslips for immunofluorescence staining (n=5) and BrdU incorporation (n=5) and in 100 mm culture dishes for RNA extraction (n=2). After 18 hours the medium was replaced with DMEM containing 10% FCS for further 24, 48 or 72 hours. BLOCK-iT Fluorescent Oligo (Invitrogen) was used as positive control of transfection efficiency. The possible off-target effect of silencing siRNAs was evaluated by measuring the activation of IFN-ß pathway using a dual-luciferase reporter assay, Western blotting for STAT1 (signal transducer and activator of transcription 1), and real-time PCR for porcine IFN-ß. The corresponding materials and methods are presented in the online data supplement.
Statistical Analysis
Results are shown as mean±SEM. Comparisons between treated and control groups were analyzed by Student t test. Differences were considered statistically significant at values of P<0.05.
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Results |
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S-SMCs and R-SMCs Isolated From Porcine CA Exhibit Distinct Protein Pattern
The relative volume of spots, which is directly related to the concentration of the corresponding protein,3,13 was computed in the pH range of 4.5 to 8.5. We quantified 1262±47 spots in S-SMC gels (Figure 1A) and 1249±49 spots in R-SMC gels (Figure 1B). Pairwise spot matching reached at least 75% in both groups. Synthetic gels built with 3 gels of each phenotype contained 744 spots for S-SMCs (Figure 1C) and 870 spots for R-SMCs (Figure 1D); an arbitrary number was assigned to each spot. Seven differentially expressed spots were found; their features are described in supplemental Table II available at http://circres.ahajournals.org and their location is indicated by arrowheads on Figure 1C and 1D. Three of the 7 differentially expressed proteins had a spot relative volume significantly superior in S-SMCs than in R-SMCs and 4 had a spot relative volume significantly superior in R-SMCs than in S-SMCs.
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S100A4 Is Predominantly Expressed in R-SMCs
Among the 7 spots of interest, the most differentially expressed (arrowheads on Figure 1A and 1B) was the spot 2697 (Figure 1C and 1D), which exhibited the higher relative volume in R-SMC gels than in S-SMC gels (Figure 1E and supplemental Table II in the online data supplement). The set of peptides obtained by MS/MS for this spot was compared with the combined Swiss-Prot and TrEMBL databank. The first matching proteins were bovine S100A4 (Accession No. P35466), which weights 11668 Da, and human S100A4 (Accession No. P26447), which weights 11721 Da. For both proteins the matching consisted of 6 peptide matches with a total Mascot score of 191. S100A4 is also known as metastasin, mts1 protein, placental calcium-binding protein and calvasculin. The molecular weight (12.3 kDa) and isoelectric point (5.59) calculated on 2D-gels were in agreement with those calculated on 2D-maps for HeLa cells and human lymphocytes.
The monoclonal anti-S100A4 was characterized as being an IgM. This antibody as well as the commercially available polyclonal anti-S100A4 confirmed by means of Western blot analysis the high expression of S100A4 in R-SMCs compared with S-SMCs (Figure 2A and 2B). A single band was detected at the level of 12.3 kDa as expected for S100A4 protein. The presence of a single spot at the expected location of S100A4 on 2D-gel immunoblot (Figure 2C) validated the specificity of the S100A4 monoclonal antibody.
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S100A4 mRNA expression normalized to GAPDH mRNA expression was 25.6 times higher in R-SMCs compared with S-SMCs (P<0.001, Figure 2D). Amplified S100A4 and GAPDH mRNA appeared at expected locations on agarose gels ie, 228 and 129 kb, respectively (Figure 2E).
Double immunofluorescence staining of resting SMCs using the monoclonal anti-S100A4 and anti--SMA showed that the proportion of -SMA expressing cells was 99.9±0.1% for S-SMCs and 89.9±1.5% for R-SMCs, with a lower intensity in R-SMCs.2 S100A4-positive S-SMCs represented 13.6±3.7%, all of them were also positive for -SMA. In contrast, the proportion of S100A4-positive R-SMCs represented 82.3±5.6% (P<0.001 compared with S-SMCs), among them 73.7±4.7% also expressed -SMA. R-SMCs exhibited a strong diffuse cytoplasmic staining; moreover in several of them the staining was located along some stress-fibers (Figure 3A). Double staining of S100A4 and -SMA confirmed their coexpression in stress fibers. The rare S100A4-positive S-SMCs exhibited the same staining pattern than R-SMCs.
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S100A4 Expression Is Enhanced During Cell Migration
We have demonstrated using an "in vitro wound" model that R-SMCs exhibit a higher migratory activity than S-SMCs.2 This increased motility was associated to an increased immunofluorescence staining of S100A4 18 hours after wounding (Figure 3B, b). The pattern of expression was more diffuse in migrating cells (Figure 3B, c) compared with quiescent cells (Figure 3A, b), possibly because of the decreased number of stress fibers in these cells. S-SMCs practically did not migrate during the same time interval and the staining of S100A4 remained low in these cultures (Figure 3B, a).
S100A4 Is Involved in Cell Proliferation
Immunofluorescence staining showed that both silencing siRNAs, P186 and P328, downregulated the number of S100A4-positive R-SMCs 24 and 48 hours after transfection compared with the scramble siRNA (Figure 4A). The silencing effect of siRNAs was maximal at 48 hours. Western blotting further showed that P186 and to a lesser extent P328 siRNAs decreased S100A4 expression in R-SMCs compared with the scramble siRNA at both time points studied (Figure 4B). Western blotting using total actin antibody showed that loading was similar in all conditions (Figure 4B). This was associated to a decrease of S100A4 mRNA expression, again more pronounced with P186 siRNA (Figure 4C). Both silencing siRNAs induced a decreased BrdU incorporation at 24 hours, which was maintained at 48 hours (Figure 4D) indicating that S100A4 downregulation is associated to reduced R-SMC proliferation. This effect was no more observed at 72 hours (data not shown), showing that treated cells recovered their basal level of proliferation. In all situations, no significant differences were observed between untreated cells and cells transfected with scramble siRNA. None of the silencing siRNAs activated antiviral mechanisms such as IFN-ß pathway (see supplemental Figure I).
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S100A4 Expression Is Modulated by Growth Factors and Coculture With ECs
Western blotting showed that the level of expression of S100A4 in R-SMCs was slightly decreased when cells were exposed to SFM as compared with 10% FCS (Figure 5). We have reported that S-SMCs modulate to R-phenotype after treatment with PDGF-BB, FGF-2 or coculture with ECs.2 As expected, after these treatments the proliferation of both SMC phenotypes was increased,2 moreover, S100A4 expression was markedly upregulated (Figure 5); coculture with ECs had the most pronounced effect.
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When SMCs were cultured in the presence of heparin or TGF-ß2 (Figure 5), both phenotypes grew more slowly than their controls,2 and the expression of S100A4 was significantly reduced in R-SMCs, particularly after heparin treatment.
S100A4 Is Expressed in SMCs of IT Induced After Stent Implantation in Porcine CA
Immunohistochemistry (Figure 6A) performed with the monoclonal anti-S100A4 showed that S100A4 was not detectable in the EC layer, media and adventitia of healthy porcine CA (Figure 6A, a). Ten days after stent implantation, S100A4 was strongly upregulated in the IT (Figure 6A, b). The underlying media remained almost negative. Although the IT cellularity decreased at 30 days,8 S100A4 expression remained appreciable in IT (Figure 6A, c).
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Double immunofluorescence staining (Figure 6B) performed 10 and 30 days after stent implantation with S100A4 and either -SMA, smoothelin or CD-172 (identifying porcine macrophages) antibodies confirmed that S100A4 was absent from the media and present in IT. As expected, the media was strongly positive for -SMA (Figure 6B, a) and smoothelin (Figure 6B, b). At 10 days, the IT was positive for -SMA and practically all -SMA-positive cells were strongly labeled with the anti-S100A4 (Figure 6B, a). Few cells expressed smoothelin and among them some were positive for S100A4 (Figure 6B, b). At 30 days, many -SMA-positive cells were still positive for S100A4 (Figure 6B, d); a small proportion of them was also positive for smoothelin (Figure 6B, e), which was as expected reexpressed in many intimal SMCs compared with 10-day-old lesions. At both time points, CD-172-positive cells were practically always negative for S100A4 (Figure 6B, c and f).
Double immunofluorescence staining performed early (7 and 10 days) after stent implantation with anti-S100A4 and anti--SMA demonstrated that S100A4 was expressed in few medial SMCs (supplemental Figure II).
S100A4 Is Expressed in Human Atherosclerotic and Restenotic Lesions
After immunohistochemistry (Figure 7) in human specimens, S100A4 was detectable in the IT present in the CA of children (Figure 7a) and young adults, in the fibrous cap of atheromatous plaques (Figure 7c) and in restenotic lesions (Figure 7e) of adult CA. -SMA staining (Figure 7b, 7d, and 7f) performed on adjacent sections showed that S100A4-positive cells were SMCs. Few medial SMCs expressed S100A4.
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Discussion |
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Following a strategy that has been successful in the rat model,3 we have identified by means of 2D-PAGE analysis differentially expressed proteins in 2 phenotypically distinct porcine CA SMC populations, which are recovered in different proportions from the media and the experimental IT after stent implantation.2 2D-PAGE analysis has the advantage of revealing differentially expressed major cellular components; in addition, improvement of MS technique14 has facilitated protein characterization. We have selected S100A4 that is predominantly expressed in R-SMCs and, using a newly produced monoclonal antibody, shown that it is absent in the normal media and highly expressed in experimental IT 10 to 30 days after stent implantation; moreover S100A4 is found in few medial SMCs as early as 7 days after injury. This protein is expressed in a majority of SMCs in human CA atheromatous plaque and restenotic lesion, thus representing a new marker of porcine and human arterial SMCs implicated in these processes. It should be noted that in our porcine model intimal macrophages do not express S100A4, whereas human monocyte/macrophages have been shown to express it.15,16 This discrepancy may result from interspecies differences or to macrophage phenotypic heterogeneity and/or level of differentiation. Cellular retinol binding protein-1, previously identified as marker of rat epithelioid SMCs in vitro and of intimal SMCs in experimentally induced IT,3,4 has not been proven relevant for the study of human lesions.1 Among several genes identified, mainly in rodents, as differentially expressed in distinct SMC phenotypes osteopontin is the only one to be relevant to human lesions where it is related to atheromatous plaque calcification.17 Our results reinforce the assumption that the pig is a valuable model for human atheromatous lesions although there are well established differences between the phenotypic features of human plaques18 and those induced experimentally in the porcine CA.8,19
To date, 5 S100 family members have been described in SMCs: S100A1, associated with stress fiber-like structures,20 S100A2, specifically expressed in the nucleus,20 as well as S100A6,21 S100A1021 and S100A1320 whose localization is at present not determined. As the C-terminal sequence of S100 proteins is not conserved among isoforms, it provides a mean to design specific antibodies for each S100 protein.22
The S100A4/mts1 gene was initially shown to be upregulated in highly metastatic mouse mammary adenocarcinoma cells versus non metastatic ones23 and to confer a metastatic phenotype to tumor cells.24–27 We have demonstrated that R-SMCs proliferate and migrate more actively than S-SMCs.2 In the present study we show that increased S100A4 expression is associated with upregulation of R-SMC proliferative and migratory activities. Moreover, siRNA experiments demonstrating that S100A4 downregulation is associated with reduced cell replication are compatible with a functional role of this protein.
The localization of S100A4 in -SMA-positive stress fibers of quiescent R-SMCs is in agreement with data showing its interaction with various cytoskeletal proteins, such as nonmuscle myosin heavy chains,28–30 actin,31 and nonmuscle tropomyosin.15 There is increasing evidence that S100A4 is constitutively secreted by tumor cells and tumor-activated stromal cells32 and acts as angiogenic factor33 thereby promoting tumor progression through plasminogen and metalloproteinase activation and extracellular matrix protein degradation.32,34,35 It is conceivable, albeit not proven, that a subset of SMCs participate in migration promotion and IT development by secreting S100A4.
The receptor for advanced glycation end products (RAGE) is the putative receptor for several S100 proteins, including S100A4,36 although no direct evidence is available for S100A4/RAGE interaction. RAGE has been shown to be upregulated at sites of accelerated vascular lesions in diabetic mice37 and to be involved in neointimal formation after balloon-injury in a mouse model.37 We show that S100A4 is upregulated in SMCs of porcine CA IT, and of IT, atheromatous plaque and restenotic lesions in humans; additional work is necessary to investigate whether S100A4/RAGE interaction plays a role in the pathogenesis of these lesions. In this respect, it has been shown that serotonin induces S100A4 release by SMCs isolated from human pulmonary artery, which, in turn, could bind to RAGE thus leading to cell proliferation and migration.38
SMC phenotypic modulation is a landmark for plaque development and restenosis.1 Our previous work has demonstrated that treatment with PDGF-BB, FGF-2 and coculture with ECs promote porcine S-SMC proliferation and modulation into R-SMCs; if the stimulus ceases, they return to the original phenotype.2 We show here that this modulation is associated with S100A4 expression. However, decreasing R-SMC proliferation by heparin or TGF-ß2 has no influence on their morphology2 and does not switch off S100A4 expression. Identification of factor(s) interfering with S100A4 expression in R-SMCs would be important for the understanding of their differentiation mechanisms and may be useful to influence the evolution of IT or atheromatous changes.
We have recently shown that SMCs of human CA lesions, such as erosions and restenotic plaques, modulate toward the myofibroblastic phenotype.18 The neo-expression of S100A4 in these lesions may represent a marker of SMC/myofibroblast modulation.
In conclusion, we show that S100A4 is a marker of porcine R-SMCs in vitro and of intimal SMCs in vivo, both in pig and man. Further work concerning its mechanisms of expression and secretion, and of its interaction with possible receptors, such as RAGE, should be useful to better understand the evolution of atherosclerosis and restenosis processes.
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Acknowledgments |
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We thank Philippe Henchoz, Giuseppe Celetta, and Anita Hiltbrüner-Maurer for their excellent technical assistance; Sophie Clément for helpful discussion and Jean-Claude Rumbeli for photographic work. We are grateful to Dominique Garcin and Jean-Baptiste Marq for their valuable advice and tools needed for the IFN-ß pathway study.
Sources of Funding
This study was supported by the Swiss National Science Foundation, grant No. 32–068034.02. Hiroyuki Hao was supported by the Grant-in-Aid for researchers, Hyogo College of Medicine.
Disclosures
None.
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Footnotes |
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Original received February 24, 2006; first resubmission received August 21, 2006; second resubmission received December 27, 2006; revised second resubmission received February 6, 2007; accepted February 22, 2007.
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