Angiotensin II Receptor Blocker Inhibits Neointimal Hyperplasia Through Regulation of Smooth Muscle–Like Progenitor Cells
Objectives— Angiotensin II (ATII) type 1 receptor (AT1R) blocker (ARB) has been shown to inhibit neointimal formation. Bone marrow–derived mononuclear cells (BM-MNCs) give rise to smooth muscle (SM)-like cells at injured arterial wall and contribute to neointimal formation. However, role of the renin—angiotensin system in the homing process of SM-like cells during neointimal formation is unknown.
Material and Methods— When human BM-MNCs and peripheral blood MNCs (PB-MNCs) were cultured under treatment with PDGF-BB and bFGF, these cells gave rise to SM-like cells with expression of αSMA, SMemb, and SM1 proteins. RT-PCR showed the expression of AT1R, ATII type 2 receptor (AT2R), αSMA, and SMemb mRNAs. ATII accelerated the differentiation of SM-like cells, which was inhibited by an ARB CV11974 (P<0.05). We then examined the effects of ATII, CV11974, and AT2R antagonist PD123319 on neointimal formation and BM-derived SM-like cell incorporation at injured arteries in vivo. BM from green fluorescence protein (GFP)-transgenic mice was transplanted to irradiated WT mice. GFP-BM chimera mice were subjected to wire injury on the left femoral artery. ATII (100 ng/kg/min) stimulated whereas CV11974 (1 mg/kg/d) inhibited neointimal formation. Number of GFP+αSMA+ cells at neointima correlated with the intima/media ratio (r=0.69, P<0.05).
Conclusion— BM-derived SM-like progenitor cells contributed to the neointimal formation after arterial injury. ATII accelerated whereas ARB suppressed this process. These are new aspects of the ARB-mediated inhibition of atherosclerotic disease progression.
It has been established that the renin-angiotensin system (RAS) contributes to not only hypertension but also vascular proliferative disorders such as atherosclerosis.1,2 Previous studies indicated that not only the systemic RAS but also the tissue RAS have significant effects on atherosclerosis development and neointimal hyperplasia after angioplasty.3,4 ATII produced within vascular tissues activates ATII type 1 receptor (AT1R), leading to accumulation of inflammatory cells, fibrosis, and proliferation or migration of vascular smooth muscle cells (VSMCs).5–9 These effects of tissue ATII further promote atherosclerosis progression. In fact, either angiotensin converting enzyme inhibitors (ACEIs) or AT1R blockers (ARBs) have been shown to inhibit ATII-mediated endothelial dysfunction and atherosclerosis.10,11 Large clinical trials such as HOPE and LIFE studies also demonstrated similar efficacies.12,13 Especially ARB has a strong inhibitory efficacy on vascular remodeling and neointimal hyperplasia after vascular injury. Indeed, we and others have shown that ordinary dose of ARB effectively suppresses restenosis and cardiovascular events after percutaneous coronary intervension (PCI).14–17
Development of atherosclerosis had been considered to occur as a consequence of the degenerative and inflammatory processes in the vascular wall.18 However, we and others previously demonstrated that bone marrow (BM)-derived MNCs could differentiate into endothelial-like cells and smooth muscle (SM)-like cells at injured vessels.19–21 The former is called endothelial progenitor cells (EPCs), and the latter is called SM-like progenitor cells.19–21 Noteworthy, these BM-derived SM-like cells are incorporated into neointima after severe but not mild arterial injury.22,23 Therefore, neointimal hyperplasia would be mediated not only by the migration or proliferation of preexisting VSMCs in the media but also by the homing of BM-derived SM-like progenitor cells. However, role of the RAS on BM-derived SM-like progenitor cells during the development of atherosclerosis are little understood. Accordingly, here we investigated whether BM- or PB-MNCs could differentiate into SM-like cells in vitro and in vivo. We further examined the effects of an ARB CV11974 and ATII type 2 receptor (AT2R) antagonist on SM-like cell mobilization and incorporation into wire-injured arterial tissue with or without infusion of ATII employing green fluorescence protein (GFP)-BM chimera mice model.
Culture of Smooth Muscle–Like Cells
The ethical committee of Nagoya University School of Medicine approved all the experimental protocols. Human BM-MNCs were collected from unused cells barely left in blood bags used for collecting BM cells during the treatment of critical limb ischemia according to the Nagoya-TACT protocol as previously reported.24 Human PB-MNCs were isolated from healthy volunteers. BM- or PB-MNCs (5.5×105cells per cm2) were cultured on fibronectin-coated plastic dishes in HuMedia-SG2 (KURABO) supplemented with platelet-derived growth factor (PDGF-BB: 10 ng/mL) and basic fibroblast growth factor (bFGF: 10 ng/mL).25 Human aortic SMCs (HASMCs, C2571 TAKARA) were also cultured in HuMedia-SG2. HASMCs were seeded at a density of 2500 cells per cm2. At days 4, 8, and 12 of culture, medium was changed and nonadherent cells were removed. We examined the effects of ATII (0.001, 0.1, 10 nmol/L) and CV11974 (0.1, 10, 1000 nmol/L) on these cells during culture. At day 14, the number of cells of 4 different high-power fields in each well was counted and expressed as cells/HPF.
At day 14 adherent cells were fixed with 4% paraformaldehyde and permealized with 0.2% TritonX-100. After blocking with 10% human albumin, cells were incubated with monoclonal antibodies (MAbs) for CD31 (Clone JC/70A, Dako), α-smooth muscle actin (αSMA) (Clone 1A4, SIGMA), SMemb (Clone 3H2,YAMASA), and SM1 (Clone 3F8, YAMASA). Bindings of primary MAbs to cells were detected with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibody (ICN). Nuclear staining with DAPI (4′,6-diamidino-2-phenylindole, Sigma) was performed to determine the proportion of SM-like cells per adherent cells after 14 days of cultivation by manually counting DAPI-stained pyknotic nuclei.
Western blotting was performed to identify the expression of vascular SMC–specific cytoskeletal proteins. In brief, cells were homogenized in lysis buffer containing 500 mmol/L Tris HCl (pH 6.8), 10% SDS, and Glycerol. The protein contents of the lysate were determined by DC protein assay kit (Bio-Rad), and equal amounts of protein were denatured by boiling, reduced in SDS, followed by SDS-polyacrylamide gel electrophoresis (PAGE) in 12.5% separating gel and 3.0% stacking gel. The proteins were transferred to polyvinylidene fluoride (PVDF) membrane and immunoblotted using MAbs to β-actin (Sigma), αSMA, SMemb, and SM1 at dilutions of 1:500. Secondary anti-mouse antibodies conjugated to horseradish peroxidase (Amersham) at a 1:5000 dilution were used for detection by chemoluminescence (ECL plus Western Blotting Detection system, Amersham) and X-ray film exposure (Kodak). HASMCs were used as positive control cells for SM–specific markers.
We extracted total RNA from freshly isolated PB-MNCs, SM-like cells, and HASMCs. We prepared cDNA from total RNA adjusted by TRIzol reagent (Invitrogen). Reverse transcription was performed with 1 μg of total RNA. PCR primers were as follows: GAPDH 5′-CTTCACCACCATGGAGGAGG-3′and 5′-TGAAGTCAGAG-GAGACCACC-3′(557bp), αSMA 5′-ACTGCCTTGGTGTGT-GACAA-3′ and 5′-TGGTGCCAGATCTTTTCCATG-3′ (248bp), SMemb 5′-ACACACTGAACGCCGAGCTAG-3′ and 5′-CTAATTTGTTGGCGGCTGCTC-3′(231bp), AT1R 5′-GATGATTGTCCCAAAGCTGG-3′ and 5′-TAGGTAATTGCCAAAGGGCC-3′ (255bp), AT2R 5′-TTCCCTTCCATGTTCTGACC-3′ and5′-AAACACACTGCGGAGCTTCT-3′ (191bp).
PCR reactions were initiated with denaturing incubation at 95°C for 5 minutes (GAPDH, αSMA, and SMemb) and for 2 minutes (AT1R and AT2R), and finished with extension incubation at 72°C for 5 minutes (GAPDH, αSMA, and SMemb) and for 2 minutes (AT1R and AT2R). The number of cycles was 25 for GAPDH and 30 for αSMA and SMemb. Cycles were performed as follows: 30 sec at 95°C, 30 sec of annealing at 58°C, and 30 sec of extension at 72°C. The number of cycles was 35 for AT1R and 45 for AT2R. Cycles were performed as follows: 30 sec at 94°C, 1 minute of annealing at 58°C, and 1 minute of extension at 72°C. After agarose gel (1.5%) electrophoresis in the presence of ethidium bromide, the PCR products were revealed by ultraviolet irradiation.
SM-like cells were detached using 1mmol/L EDTA in PBS(−) (pH 7.4), harvested by centrifugation, and resuspended in 500 μL of the culture medium, counted and placed in the upper chamber of a modified Boyden chamber. The chamber was placed in a 24-well culture dish containing the culture medium with or without ATII (10 ng/mL) and/or PDGF-BB (50 ng/mL). After 3 hours of incubation at 37°C, the lower side of the filter was washed with PBS(−) and fixed with 2% paraformardehyde. Migrated cells into the lower chamber were counted manually in randomly selected 3 microscopic fields.
All experimental protocols were performed in accordance with the guidelines for animal research of Nagoya University School of Medicine. GFP-transgenic mice with C57BL/6J background were kindly provided by Dr M Okabe (Osaka University, Japan).26
BM cells were obtained by flushing the tibias and femurs of 10-week-old male mice. MNCs were isolated from either BM or peripheral blood by density centrifugation over Histopaque-1083 (Sigma Diagnostics Inc, USA) and then washed in PBS. SM-like cells were obtained with the same protocol as adopted to obtain human SM-like cells.
Bone Marrow Transplantation and Chimera Mice Production
Ten-week-old male C57BL/6J mice were subjected to lethal irradiation (10Gy) (MBR-1520R, HITACHI). Each irradiated recipient received 5×106 BM cells extracted from GFP-transgenic mice by tail vein injection. On day 27 after bone marrow transplantation (BMT), the recipient mice were minimally phlebotomized at the tail, and circulating leukocytes were checked for the expression of GFP by flow cytometry.
Femoral Artery Injury Model
Four weeks after BMT, wire injury operation was performed using a dissecting microscope (SZ61-C-SET, Olympus). We performed transluminal mechanical injuries of femoral arteries as described previously.27 In brief, mice were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The straight spring angioplasty wire (0.36 mm diameter, Boston Scientific) was introduced into the left iliac artery via femoral artery. Subpressor dose of ATII (100 ng/kg/min, n=5), CV11974 (1 mg/kg/d, n=5), ATII+CV11974 (n=5), ATII type 2 receptor antagonist PD123319 (30 mg/ day, n=5) or all (n=5) were administered continuously using osmotic minipumps (model 2004, Alzet Corp) implanted subcutaneously into the mice for 4 weeks. To monitor hemodynamic changes after arterial injury, arterial blood pressure was measured under conscious states by tail-cuff method using a BP-98A (Softron).
At 4 weeks after injury, femoral arteries were harvested under deep anesthesia. To investigate the degree of neointimal formation and the involvement of BM-derived cells into wire-injury–induced vascular remodeling lesions, the proximal segments of the injured arteries were fixed in paraformaldehyde, embedded in paraffin, sectioned at 4 μm thickness, and stained with Elastica van Gieson. Neointima and media areas were measured by a computer-assisted image analyzer using Image J Software (BioArts). The distal segments of the injured arteries were snap-frozen in Optimal Cutting Temperature (OCT) compound (Sakura Finetek). Cross sections (5 μm) were incubated with Cy3-conjugated anti-αSMA antibody (Sigma) and rat anti-SM1 MAb (KM3669), followed by Alexa-Fluoro-conjugated anti–rat IgG antibody. After immunofluorescence staining, nuclei were counterstained with DAPI (Molecular Probes). Five different sections were used from each mouse to qualify the femoral artery lumen and were observed under a microscope equipped with mercury/halogen dual-illumination system and a charge-coupled device (CCD) camera (BZ-8000, Keyence). High-resolution fluorescence photo images of total fields were taken from each section.
All values are expressed as means±SEM. For continuous variables, 1-way ANOVA followed by Scheffe multiple comparison test were performed with Statview software (version 5.0, Abacus Concepts). A value of P<0.05 was regarded as statistically significant.
Phenotype of BM- and PB-MNC–Derived SM-Like Progenitor Cells in Mice and Human
Either human BM- or PB-MNCs were cultured in fibronectin-coated dishes in the HuMedia-SG2 medium supplemented with PDGF-BB and bFGF. Adherent cells that consisted of heterogeneous cell types with round or spindle shapes were observed after 4 or 5 days of cultivation. By day 14, polygonal and stellate-shaped cells became dominant, and these cells were stained positive for αSMA, SMemb, and SM1 but not CD31 (supplemental Figure IA, available online at http://atvb.ahajournals.org). SM-like cells derived from mice BM- or PB-MNCs looked similar in shape to those of human SM-like cells. Mice SM-like cells were also positive for αSMA, SMemb, and SM1 but not CD31 (supplemental Figure IA). To further confirm the presence of smooth muscle–specific proteins in these cells, cell lysates were subjected to Western blot analysis. These cells expressed αSMA, SMemb, and SM1 proteins (supplemental Figure IB). RT-PCR analysis further revealed that these cells expressed not only αSMA and SMemb but also AT1R and AT2R mRNAs (supplemental Figure IC).
Effects of ATII and CV11974 on SM-Like Cells
The fact that BM- or PB-MNC-derived SM-like cells express AT1R prompted us to investigate the effects of ATII, CV11974, or both on these cells. Addition of ATII significantly increased the number of SM-like cells that were positive for αSMA in a dose-dependent manner. In contrast, CV11974 alone decreased the number of SM-like cells during 14 days culture to the level even lower than that observed in nontreated control cells (supplemental Figure IIA). And ATII-induced αSMA+ SM-like cells differentiation was significantly inhibited by CV11974 (αSMA+cells/HPF: ATII, 112.9±8.2; ATII+CV11974, 62.6±4.0; CV11974, 32.5±2.2; control, 63.2±4.4, *P<0.05, **P<0.001; versus control, †P<0.05, ††P<0.001; supplemental Figure IIB). Furthermore, cell migration assay revealed that ATII enhanced migratory activity of SM-like cells, and the migration was significantly augmented in the presence of PDGF-BB (SM-like cells/HPF: control, 39.5±2.5; ATII, 82.1±4.8; ATII+control, 114.7±6.5, **P<0.001; versus control, †P<0.05; supplemental Figure IIC).
Effects of ATII, CV11974, and PD123319 on Neointima Formation After Vascular Injury
Next, we examined the effects of ATII, CV11974, and PD123319 on recruitment of BM-derived SM-like cells to neointima formed after arterial injury using GFP-BM chimeria mice. As we have reported previously, this vascular injury model induces reproducible neointimal hyperplasia. We first confirmed reconstitution of the hematopoietic system by checking the expression of GFP on circulating blood cells by flow cytometry at 4 weeks after BMT. We found that most of the circulating leukocytes were positive for GFP in BM-GFP transplanted chimera mice (89.3±0.4%, n=36). This indicates that most of the circulating blood cells were derived from replaced donor-derived GFP-positive BM cells. Then, the left femoral arteries of GFP-BM chimera mice were injured by inserting a straight spring guide wire. After the vascular injury, osmotic minipumps were implanted subcutaneously. Systemic blood pressure of each group (control PBS, ATII, CV11974, ATII+CV11974, PD123319, and ATII+CV11974+PD123319 group) did not differ significantly among the groups (data not shown). Four weeks after vascular injury, the injured femoral arteries were examined histologically (Figure 1A). Compared with control nontreated mice, neointimal formation was markedly enhanced by ATII infusion, whereas this was reduced by CV11974 treatment. Consistent with our in vitro studies, mice administered with CV11974 alone revealed a significantly reduced neointimal hyperplasia compared with control mice administered with PBS. However, PD123319 administration did not affect neointimal hyperplasia (intima/media ratio: ATII, 3.02±0.17; ATII+CV11974, 2.26±0.13; CV11974, 1.29±0.07; PD123319, 2.16±0.13; ATII+CV11974+PD123319, 2.35±0.13; control, 2.13±0.13, *P<0.05; versus control, †P<0.05, ††P<0.001; Figure 1B).
CV11974 Decreased BM-Derived SM-Like Cells Infiltration Into Arterial Walls and Reduced Neointima Formation
To elucidate the involvement of BM-derived SM-like cells in vascular remodeling and neointimal formation after vascular injury, we examined wire-injured femoral arteries under fluorescence microscopy. Most cells in the neointima lesion were positive for αSMA (Figure 2A). GFP-positive cells were also detected not only in neointima lesion but also in the media and adventitia at the injured arterial wall (Figure 2B). Furthermore, abundant GFP-positive cells costained with αSMA were observed at all the portions of neointima, media, and adventitia of the injured arteries (Figure 2C through 2G). The number of αSMA+, GFP+, and αSMA+GFP+ cells were significantly increased by ATII administration, whereas these were reduced by CV11974 infusion. The results of the number of αSMA+, GFP+, and αSMA+GFP+ cells paralleled with the results of the intima/media ratio (Figure 2H through 2J). The number of BM-derived GFP+αSMA+ cells in the neointima was significantly correlated with the intima/media ratio (r=0.69, P<0.05; Figure 3). The cells that were positive for both GFP and SM1 (a marker of relatively mature SMCs) were hardly detected in the neointima in any groups (data not shown). These findings indicate that BM-derived αSMA positive cells might not become mature SMCs at least 4 weeks after the wire-injured artery model.
Major findings of the present study are as follows: (1) Both mice and human BM- or PB-MNCs, when cultured in the presence of bFGF and PDGF-BB in vitro, differentiated into SM-like progenitor cells with expressions of αSMA, SMemb, and SM1. (2) Human SM-like cells also expressed both AT1R and AT2R. (3) The number of differentiated human SM-like cells significantly increased in response to ATII, and this was suppressed by cotreatment with CV11974 to the level similar to the nontreated control group. Interestingly, addition of CV11974 alone without ATII also suppressed the number of SM-like cells to the level lower than that of the control group. (4) ATII stimulated the neointimal formation in wire-injured femoral arteries in mice as compared with nontreated control animals, and this was suppressed by coadministration of CV11974. Like in vitro findings, as compared with nontreated animals CV11974 alone reduced the neointimal formation in mice without ATII administration. AT2R blocker PD123319 did not affect the neointimal formation. (5) BM-derived αSMA-positive SM-like cells (GFP+αSMA+ cells) were found transmurally at neointima, media, and adventitia regions at the injured arteries, and ATII significantly enhanced the incorporation of GFP+αSMA+ cells at injured arterial walls, and this was again suppressed by CV11974 treatment. The latter in vivo results suggest that ATII not only stimulates neointimal hyperplasia but also stimulates BM-derived SM-like progenitor cell incorporation after wire injury. This was confirmed that AT1R blocker CV11974 significantly inhibited both of ATII-induced neointimal formation and BM-derived SM-like cell incorporation.
Previous studies have indicated that ATII promotes atherosclerosis through vasoconstriction, formation of reactive oxygen species, proliferation and migration of vascular smooth muscle cells (VSMCs), and other numerous mechanisms.2,28,29 Until recently, the main mechanisms of atherosclerotic development had been considered exclusively as a consequence of migration or proliferation of mature synthetic VSMCs from the media of arteries.18 However, we and others have recently demonstrated that not only migration and proliferation of medial VSMCs but also homing of circulating BM-derived SM-like progenitor cells contribute to the development of atherosclerosis.20,21,30,31 Particularly, it has been experimentally confirmed that significant number of vascular wall composing cells in neointima and media originated from BM-derived SM-like progenitor cells in severely injured arteries.21–23 However, it was little known as to whether the RAS plays significant roles in the differentiation of SM-like progenitor cells from BM-MNCs and incorporation of these cells to neointimal formation in vivo.
In the present study, we found that ATII contributes to the vascular neointimal formation by promoting accumulation of BM-derived SM-like cells in mice with the wire-mediated severe endovascular injury model. In the GFP-BM chimera mice the number of incorporated SM-like cells was significantly increased by ATII administration, and this was blocked by CV11974. Our in vitro culture experiments also showed that CV11974 inhibited phenotypic changes from BM- or PB-MNCs to SM-like progenitor cells. Importantly, in the present study, there was a positive and significant correlation between the number of accumulated BM-derived SM-like cells and the intima/media ratio of the wire-injured arteries. These results collectively suggest that the ARB CV11974 has an antiatherosclerotic effect not only by inhibiting the proliferation/migration of medial VSMCs but also by inhibiting differentiation and accumulation of BM-derived SM-like progenitor cells at injured arteries.
Interestingly, suppression of the accumulation of SM-like progenitor cells by CV11974 was also observed even without ATII administration. This finding indicates that CV11974 might inhibit the function of locally produced ATII by the tissue RAS and suppress the accumulation of SM-like progenitor cells at injured sites. Morishita and coworkers previously showed that the activity of the tissue RAS is significantly enhanced at the injured arterial wall.4 In such pathological states, CV11974 might have blocked locally enhanced intrinsic RAS activity and thereby inhibited neointimal formation. Alternatively, CV11974 might directly affect circulating BM-derived SM-like cell kinetics by an ATII-dependent mechanism because these cells express AT1R and AT2R on their surface in our study. In this sense, Ohtani and coworkers recently showed that an ARB valsartan inhibited in-stent restenosis by suppressing circulating SM-like progenitor cells in an animal mode with hypercholesterolemia.32
In our study, AT2R blocker PD123319 did not influence the accumulation of BM-derived SM-like cells. This indicates that the ATII-AT2R system may play a minimal role in the process of BM-derived SM-like cell incorporation and the formation of neointima after arterial injury at least in our model. The fact that CV11974 has an inhibitory effect on accumulation of SM-like cells at injured vascular walls may explain one of the mechanisms of antiatherosclerotic efficacies of ARBs in several clinical studies. Previously we reported that subpressor dose of CV11974 reduced in-stent neointimal hyperplasia and cardiovascular events after PCI in humans.14,15 The Val-Prest and VALVACE studies also showed that an ARB valsartan significantly inhibited the occurrence of restenosis after coronary artery stenting in complexed type B2 or C lesions.16,17 The current experimental study may account for one of the potential mechanisms of the results of those previous clinical studies.
There are several limitations in the present study. First, mice and human PB-MNC–derived SM-like cells in vitro were positive for both αSMA and SM1 and therefore had relatively differentiated SM phenotype. On the other hand, BM-derived SM-like cells in the injured arteries in mice vivo model were positive for αSMA but not for SM1. This indicates that SM-like cells in neointima have relatively immature SM-like phenotype but not mature contractile SM-like features. Therefore, BM-derived SM-like cells in vivo may represent less differentiated phenotype as compared with SM-like cells observed in vitro. Second, these effects were likely mediated by blocking AT1R function rather than stimulating AT2R in our model because AT2R blocker PD123319 affected neither neointimal formation nor BM-derived SM-like cell incorporation to neointima after wire injury. Horiuchi and coworkers previously showed that AT2R stimulation reacted by counteracting AT1R-mediated signaling during neoinimal formation induced by cuff replacement using AT2R knockout mice.33 The reason for this discrepant result is unknown, but the arterial injury model used in our study was wire injury and more severely damaged arterial wall compared with cuff model. We previously demonstrated that homing of BM-derived SM-like progenitors was most aggressively observed in wire injury mode compared with other injury models.22 Thus, the efficacy of AT2R blocker might be minimal in our study. Third, it is still unclear whether ATII promotes differentiations of BM- and PB-MNCs to SM-like cells or just stimulates migrations of SM-like cells. However, to discriminate the two processes is impossible because of the continuum of the two phenomena.
In conclusion, CV11974 exerted inhibitory efficacies on neointimal hyperplasia in injured arteries. The effects were achieved at least in part by the suppression of BM-derived SM-like cell infiltration at injured arterial walls. Our results support the idea that ARB such as CV11974 has multifactorial efficacies for prevention of vascular wall injuries. Accordingly, CV11974 is one of the ideal therapeutic options for hypertension, because it may suppress atherosclerotic development at injured blood vessels in addition to the blood pressure control.
We are grateful to the Kyowa Hakko Kogyo Co (Tokyo, Japan) for providing anti-SM1 MAb and to the Takeda Pharmaceutical Company Ltd (Osaka, Japan) for providing CV-11974.
Sources of Funding
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology in Japan (#16390221, #18390232, #19659201). This work was also supported in part by the Smoking Research Foundation, the Terumo Research Foundation, the Suzuken Memorial Foundation and the Takeda Research Foundation to T.M.
Original received April 26, 2007; final version accepted August 10, 2007.
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