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(Circulation. 1999;100:861-868.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Inhibition of Neointima Hyperplasia of Mouse Vein Grafts by Locally Applied Suramin

Yanhua Hu, MD; Yiping Zou, MD; Hermann Dietrich, DVM; Georg Wick, MD; Qingbo Xu, MD, PhD

From the Institute for General and Experimental Pathology (Y.H., H.D., G.W.), University of Innsbruck Medical School, and the Institute for Biomedical Aging Research (Y.Z., G.W., Q.X.), Austrian Academy of Sciences, Innsbruck, Austria.

Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail Qingbo.Xu{at}oeaw.ac.at

	Abstract

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Background—Saphenous vein grafts are widely used for aortocoronary bypass surgery as treatment for severe atherosclerosis and often are complicated by subsequent occlusion of the graft vessel.

Methods and Results—We described a mouse model of venous bypass graft arteriosclerosis that can be effectively retarded by locally applied suramin, a growth factor receptor antagonist. Mouse isogeneic vessels of the vena cava veins pretreated with suramin were grafted end to end into the carotid arteries and enveloped with a mixture of suramin (1 mmol/L) and pluronic-127 gel. In the untreated group, vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-fold and 10-fold the original thickness in grafted veins at 4 and 8 weeks, respectively. Pluronic-127 gel alone did not influence neointima formation. Suramin treatment reduced the neointima hyperplasia 50% to 70% compared with untreated controls. Immunohistochemical studies demonstrated that a significant proliferation of vascular smooth muscle cells (SMCs) constituted neointimal lesions between 4 and 8 weeks. The majority of SMCs expressed platelet-derived growth factor (PDGF) receptors- and -ß, which were significantly reduced by suramin treatment. In vitro studies indicated that suramin completely blocked PDGF receptor activation or phosphorylation stimulated by PDGF-AB, inhibited activation of mitogen-activated protein kinase (ERK) kinases (MEK1/2) and ERK1/2, and abrogated transcription factor AP-1 DNA-binding activity.

Conclusions—Suramin inhibited SMC migration and proliferation in vivo and in vitro by blocking PDGF-initiated PDGF receptor and MAPK-AP-1 signaling. These findings indicate that locally applied suramin is effective in a mouse model of venous bypass graft arteriosclerosis.

Key Words: veins • grafting • arteriosclerosis • receptors • signal transduction

	Introduction

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Autologous vein grafts remain the only surgical alternative for many types of vascular reconstruction, but obliterative stenosis (arteriosclerosis) often follows.¹ The pathogenesis of this disease is poorly understood, and no successful clinical interventions have been identified. Recently we have established a mouse model of vein graft arteriosclerosis by grafting an autologous external jugular vein to the carotid artery or isografting the vena cava vein to the carotid artery.² In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease.² This model could be useful for studying therapeutic interventions in vein graft disease.

Vein grafts become occluded when abnormal cell proliferation in the smooth muscle layer produces extra tissue in the inner lining of the vessel, a process called neointima hyperplasia.³ Although the precise mechanism initiating such cell proliferation remains to be elucidated, accumulating evidence indicates that mechanical stress plays a crucial role.^{4 5 6} Mechanical forces stimulate smooth muscle cell (SMC)-expressing and SMC-releasing platelet-derived growth factors (PDGFs)^{7 8} and induce PDGF receptor phosphorylation or activation.^{9 10} Therefore PDGF production and receptor activation play a pivotal role in initiating SMC migration and proliferation.

The binding of the ligand dimer induces dimerization of the PDGF receptors, leading to their activation through autophosphorylation of tyrosine residues in the PDGF receptor kinase domain.¹¹ This triggers a cascade of phosphorylation events involving sequential activation of Ras, Raf, mitogen-activated protein kinase (MAPK) kinase (MEK) and, finally, extracellular regulated-protein kinases (ERKs).¹¹ ERKs phosphorylate a variety of regulatory proteins, transcription factors, and other protein kinases, including activator protein-1 (AP-1) transcription factors, which play an important role in cell migration, proliferation, and differentiation.¹¹ Suramin has been shown to be a growth factor receptor antagonist that inhibits cell proliferation.¹² In this study, we investigated the role of suramin in inhibition of venous graft arteriosclerosis by using the mouse model.

	Methods

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Mice and Vein Graft Procedure
Three-month-old male C57BL/6J mice were purchased from Charles River (Sulzfeld, Germany). The procedure used for vein grafts was similar to that described previously.² Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg body wt IP). The right common carotid artery was mobilized free from the bifurcation at the distal end toward the proximal, cut in the middle, and a cuff placed at the end. The artery was turned inside out over the cuff and ligated. The vena cava vein was harvested and grafted between the 2 ends of the carotid artery by sleeving the ends of the vein over the artery cuff and ligating them together with an 8-0 suture.

Suramin Treatment and Tissue Preparation
The vena cava segments were incubated ex vivo with RPMI 1640 medium containing 0.3 mmol/L suramin (Sigma Chemical Co) at room temperature for 20 minutes before grafting to the carotid artery. Suramin (1 mmol/L) solubilized in 0.9% NaCl at 4°C was mixed with pluronic-127 gel (20% wt/vol; Sigma). Immediately after vessel grafting, suramin-gel solutions were applied to the adventitia. One control group was treated with 20% pluronic-127 gel and another group was not treated. On contact with the tissues, the solutions gelled immediately, generating a translucent layer that enveloped the grafted vessel segment. The wounds were closed after application of the gel.

The vein grafts were harvested at 1 and 3 days and at 1, 4, and 8 weeks after surgery (3 to 8 mice at each time point per group) by cutting the implanted segments from the native vessels at the cuff end. For histological analysis, perfusion with 4% phosphate-buffered formaldehyde (pH 7.2) was performed as described previously.² For frozen section preparation, vein grafts were harvested without perfusion, immediately frozen in liquid nitrogen, and stored at -80°C.

Histology and Lesion Quantification
After fixation, the grafts were cut in the middle of the vein segments, dehydrated, and embedded in paraffin. Histological sectioning began at the center of the graft to avoid the effects of the cuff. The procedure used for lesion quantification was similar to that described previously.² Briefly, we determined the thickness of the vessel wall by measuring 4 regions of a section along a cross, recorded in microns (mean±SD). We obtained 5 cross sections by selecting the first of every 3 sections from each animal.

Immunohistochemical and Immunofluorescent Staining
The procedure used for immunohistochemical assays was similar to that described previously.¹³ Briefly, sections were labeled with a mouse monoclonal antibody against -actin (Sigma). Counterstaining with hematoxylin was performed. For double immunofluorescence staining, sections were incubated with rabbit polyclonal antibodies against PDGF receptor- or -ß (Santa Cruz Biotech) labeled with swine anti-rabbit antibodies conjugated with TRITC. Sections were then labeled with a mouse monoclonal antibody against -actin conjugated with FITC, and cell nuclei were stained with Hoechst 33258.

Cell Culture
Mouse vascular SMCs were cultivated from their aortas by use of a modified procedure of Ross.¹⁴ In short, mouse thoracic aortas were removed and washed with RPMI 1640 medium. The intima and inner two thirds of the media were carefully dissected from the vessel under an anatomic microscope, cut into pieces (1x1x0.1 mm), and planted onto a gelatin-coated (0.02%) plastic bottle (Falcon). The bottle was incubated upside down at 37°C in a humidified atmosphere of 95% air/5% CO₂ for 3 hours, and then medium supplemented with 20% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL) was slowly added. Cells were incubated at 37°C for 7 to 10 days and passaged by treatment with 0.05% trypsin/0.02% EDTA solution. Experiments were conducted on SMCs that had just achieved confluence.

Immunoprecipitation, Western Blot Analysis, and Kinase Assays
For PDGF receptor isolation and MAPK assays, serum-starved SMCs were preincubated with 0.3 mmol/L suramin at 37°C for 30 minutes, incubated with PDGF-AB (100 ng/mL; Sigma) for 8 minutes, and harvested on ice in buffer A.⁹ PDGF receptor- and ERK2 were immunoprecipitated with specific antibodies and protein G–agarose (Santa Cruz Biotech) as described previously.^{9 15} Western blot analysis was performed with sequential antibodies against phosphotyrosine (4G10; Upstate Biotech Inc) and PDGF receptor-, which were detected with the ECL Detection Kit (Amersham). ERK2 activities in the immunocomplexes were measured as described previously.^{9 15}

Gel Mobility Shift Assays
Nuclear protein preparation and gel mobility shift assays were similar to that described previously.^{9 16} Briefly, 5 µg of nuclear protein extracts was incubated with 0.5 ng of an oligonucleotide containing the AP-1 binding sequence (5'-CGCTTGATGACTCAG- CCGGAA-3') labeled with [-³²P] ATP. For the competition experiment, nuclear factor (NF)-B oligonucleotide (5'-AGTTGAGGGACTTT CCCAGGC-3') was also used. Supershift assays were performed with antibodies against c-Fos (Santa Cruz Biochem).

Migration and Proliferation Assays
For migration assays, RPMI 1640 medium containing 100 ng/mL of PDGF-BB was added to wells of a modified Boyden chemotaxis chamber (NeuroProbe). SMCs (2x10⁴/well) in 50 µL of RPMI 1640 medium in the presence or absence of suramin (0.3 mmol/L) were added to the upper chambers and incubated at 37°C for 6 hours in a cell culture incubator. SMCs on the upper side of the filter were removed, the filter was stained with Diff-Quick staining solution (Dade AG), and migrated SMCs were counted under the microscope.

For proliferation assays, SMCs (1x10⁴), cultured in 96-well plates in medium containing 10% FCS at 37°C for 24 hours, were serum-starved for 3 days. SMCs were treated with suramin for 30 minutes, and then PDGF-AB and 2% serum were added and incubated at 37°C for 24 hours. [³H]-thymidine was added 6 hours before cell harvest. Radiation activities were measured.

Statistical Analysis
Statistical analyses were performed with the Mann-Whitney U test and ANOVA, respectively. Results are given as mean (±SD). A value of P<0.05 was considered significant.

	Results

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Suramin-Inhibited Neointima Hyperplasia
Vein grafts at 4 and 8 weeks (Figure 1, B, E, and H) showed neointimal hyperplasia, that is, thickening of the vessel wall up to 10 or 20 layers of cells, and increased matrix protein accumulation. Treatment of the vein grafts with pluronic-127 gel alone did not significantly influence neointima formation (Figure 1C). Interestingly, suramin-treated vein grafts showed markedly reduced neointima lesions at 4 and 8 weeks (Figure 1, D, G, F, and I).

View larger version (97K): [in this window] [in a new window]   Figure 1. Hematoxylin and eosin–stained sections of mouse control vein and vein grafts. Mouse vena cava was removed, washed with 0.9% NaCl containing heparin (500 U/mL), incubated with PBS (B, C, E, and H) or PBS supplemented with suramin (0.3 mmol/L; D, F, G, and I) for 30 minutes, and then isografted into carotid arteries. The grafted vein segments were enveloped in 20% pluronic-127 gel (C; 50 µL/mouse) or the gel containing 1 mmol/L suramin (D, F, G, and I). Animals were killed 0 (A), 4 (B, C, D, H, and G), and 8 (E, F, and I) weeks after surgery, and the grafted tissue fragments were prepared for hematoxylin-eosin sections. Arrows indicate the control vessel wall (A) and neointima (B through I); original magnification x40 for A through F; x250 for G through I.

To statistically analyze vein graft lesions, Figure 2 summarizes data of neointima thickness measured microscopically. The thickness of the vessel wall, including neointima and media, was measured and statistically compared. No significant difference between untreated (4 and 8 weeks, n=8, respectively) and pluronic-127 gel–treated (4 and 8 weeks, n=6, respectively) groups was found. Suramin treatment (4 and 8 weeks, n=8, respectively) reduced neointimal thickness 50% to 70% compared with untreated controls (Figure 2).

View larger version (50K): [in this window] [in a new window]   Figure 2. Suramin-inhibited neointima thickness in vein isografts. The procedure for animal models and the preparation of hematoxylin and eosin–stained sections are the same as described in the legend to Figure 1. Four regions of each section along a cross were measured, and 5 sections per animal were selected. Data are mean values (±SD) of intima and media or neointima thickness obtained from 6 to 8 animals per group in each time point, that is, the average of 4 measurements per histological section of 30 to 40 sections in each column. *Significant difference between suramin-treated and untreated or gel-treated groups, P<0.05.

Suramin-Inhibited SMC Accumulation and PDGF Receptor Expression
Immunohistochemical staining with monoclonal antibodies against -actin on frozen sections demonstrated the presence of abundant SMCs in venous bypass graft lesions 4 and 8 weeks after surgery (Figure 3). No positive (red) staining was seen in vein segments stained with normal rat serum as a negative control (Figure 1A). Strong staining was observed in sections from untreated and gel-treated groups 4 and 8 weeks after surgery (Figure 3, B, D, and E). Importantly, the number of positive-stained SMCs was markedly reduced in suramin-treated vein grafts at 4 and 8 weeks (Figure 3, C and F).

View larger version (121K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining demonstrates SMCs in vein grafts. Sections derived from vein grafts at 4 (A through C) and 8 (D through F) weeks were labeled with a mouse monoclonal antibody against -actin conjugated with an alkaline phosphatase and developed with the substrate with counterstaining. Arrows indicate neointima. A large number of -actin–positive cells (red) can be seen in sections of untreated (B and E) or gel-treated (D) vein grafts, and a small portion of positive cells in suramin-treated vein grafts (C and F). Original magnification x250.

Given the primary importance of PDGF, we investigated the distribution and levels of PDGF receptor- and -ß in mouse vein grafts. At 4 or 8 weeks after implantation, high levels of PDGF receptor- and -ß were detected in neointimal cells of untreated vein grafts (Figure 4). Most neointimal SMCs of untreated or gel-treated vein grafts showed a positive staining for both receptors, and very weak staining in the suramin-treated group was observed (data not shown), indicating that PDGF receptors- and -ß play a part in the development of graft lesions.

View larger version (96K): [in this window] [in a new window]   Figure 4. Double immunofluorescence labeling of vein graft sections. Cryostat sections from mouse vein grafts 4 weeks after surgery were incubated with a mouse monoclonal antibody against -actin conjugated with FITC (A and D) for 30 minutes. After washing, sections were incubated with a polyclonal rabbit-anti-PDGF receptor- (B) and -ß (E). The reaction was visualized by anti-rabbit Ig-TRITC–conjugated swine Ig. Subsequently, sections were stained with Hoechst 33258 (1 µg/mL) (C and F). Note that most SMCs (A and D) express PDGF receptor- (B) and -ß (E). Arrows denote examples of identical cells positively stained with antibodies and DNA dye (C and F). Original magnification x250.

Suramin-Inhibited PDGF Receptor Activation
To clarify the mechanism by which suramin inhibited neointima formation in mice, mouse aortic SMCs were pretreated with suramin and incubated with PDGF-AB, and PDGF receptor- was examined by immunoprecipitation with a specific antibody against PDGF receptor- from SMCs and subsequent Western blot analysis with antiphosphotyrosine antibodies. PDGF-stimulated PDGF receptor- phosphorylation was observed as early as 8 minutes (Figure 5A). Suramin alone did not activate receptors but completely blocked PDGF receptor- phosphorylation of PDGF-treated SMCs (Figure 5A).

View larger version (38K): [in this window] [in a new window]   Figure 5. Effects of suramin on PDGF receptor phosphorylation. Mouse SMCs were incubated with suramin (0.3 mmol/L) for 30 minutes, treated with PDGF-AB (50 ng/mL) for 10 minutes, and harvested. PDGF receptor- proteins were immunoprecipitated (IP), and Western blot analysis was performed with antibodies against phosphotyrosine (PY) and PDGF receptor-, respectively. A is exemplary; B shows statistical data of PDGF receptor- phosphorylation in percentage of PDGF receptor- proteins. Note no significant difference between suramin plus PDGF-AB and untreated control.

Figure 5B summarizes data of PDGF receptor- phosphorylation in percentage of PDGF receptor- proteins as determined by quantification of optical densities from ECL photograms of 2 experiments, indicating no significant difference between suramin plus PDGF and untreated groups.

Suramin Inhibition of MEK-ERK Activation
PDGF treatment resulted in rapid MEK1/2 and ERK1/2 activation. Again, suramin completely blocked MEK1/2 and ERK1/2 phosphorylation of PDGF-treated SMCs (Figure 6, upper 2 panels) and significantly inhibited MBP phosphorylation by ERK2 (Figure 6, lower panel). These results suggest that growth factor–activated MAPK pathways can be abolished by suramin.

View larger version (62K): [in this window] [in a new window]   Figure 6. Suramin-blocked MEK-ERK activation. SMCs were serum-starved for 3 days, and suramin (0.3 mmol/L) was added to the culture and incubated for 30 minutes before PDGF-AB stimulation (50 ng/mL). Top and middle, Results of Western blot analysis for phosphorylated MEK1/2 (P-MEK1/2) and phosphorylated ERK1/2 (P-ERK); bottom, results of ERK kinase assays. ERK2 kinase activities were measured on the basis of phosphorylation of myelin basic protein (MBP) substrate. Data are exemplary of 2 similar independent experiments. S indicates FCS treatment as a positive control.

Suramin Inhibition of AP-1 Binding Activation
Figure 7A shows AP-1 activation in response to PDGF-AB, which was blocked by suramin pretreatment. Figure 7B also shows the results of gel mobility shift assays performed in the presence of unlabeled AP-1 or NF-B oligonucleotides or antibodies specific to c-Fos proteins. The PDGF-induced increase in binding activity was specific for the AP-1, because increased concentrations of unlabeled AP-1 element effectively competed for binding to the factor, whereas the NF-B–binding element did not. Addition of antibody to the binding reaction resulted in the binding complexes disappearing from the AP-1 shift species, indicating the presence of Fos proteins in the DNA-binding complexes.

View larger version (55K): [in this window] [in a new window]   Figure 7. Analysis of AP-1–binding activity in SMCs. A, Gel mobility shift assay shows that suramin inhibited AP-1 activation with the use of nuclear protein extracts from SMCs treated with PDGF-AB (50 ng/mL) for 3 hours. AP-1 indicates specific DNA–AP-1–binding complexes. B, Protein extracts obtained from PDGF-treated SMCs (3 hours) were incubated with a radiolabeled oligonucleotide containing an AP-1–binding site with no addition (-) in the presence of unlabeled AP-1 or unlabeled NF-B, oligonucleotides (20:1), or in the presence of antibody specific to c-Fos. DNA-binding complexes disappeared, indicating that complexes contain c-Fos protein. Data are exemplary of 2 similar independent experiments. S indicates FCS treatment as a positive control.

Suramin-Inhibited SMC Migration and Proliferation
Because PDGF receptor-MAPK-AP-1 signal pathways are crucial in mediating cell migration and proliferation, the effects of suramin on SMC migration and proliferation were investigated. Figure 8A indicates that suramin abolished SMC migration stimulated by PDGF-BB, which is a strong chemokine for SMCs. Concomitantly, SMC proliferation induced by PDGF-AB was inhibited by suramin in a concentration-dependent manner (Figure 8B). Concentration of 0.3 mmol/L used in pretreatment of vein segments completely abrogated SMC proliferation.

View larger version (39K): [in this window] [in a new window]   Figure 8. Suramin-inhibited SMC migration and proliferation. For migration assays, a modified Boyden chemotaxis chamber was used. A polycarbonate filter with 8-µm pores was placed between the lower and upper chambers. SMCs (2x104/well) in 50 µL of RPMI 1640 medium in the presence or absence of suramin (0.3 mmol/L) were added to the upper chambers with PDGF-BB in the lower chamber and were incubated at 37°C for 6 hours. SMCs on the lower side of the filter were stained with a quick stain kit and counted under the microscope. For proliferation assays, SMCs (1x104) cultured in a 96-well plate in medium containing 10% FCS at 37°C for 24 hours were serum-starved for 3 days, treated with suramin for 30 minutes with PDGF-AB added, and incubated at 37°C for 24 hours. [3H]-thymidine was added 6 hours before cells were harvested. Radiation activities were measured. Data are mean±SD of 3 experiments. *Significant difference from other groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Recently we established and characterized a new model for the study of neointima formation of venous bypass grafts in mice.² In the current study, we have demonstrated that this mouse model is useful for investigation of effects of locally applied agents on graft disease. When vein isografts were treated ex vivo and in vivo with suramin, intimal lesions were reduced up to 70% compared with untreated controls. The mechanism of suramin-inhibited neointima hyperplasia mainly involves inhibition of SMC migration and proliferation by blocking the PDGF receptor–MAPK–AP-1 signal pathways. Such treatment might be also applicable for bypass patients, based on following reasons: First, locally applied suramin may have similar effects on inhibition of vein graft–induced lesions in humans as seen in mice, because suramin can also inhibit proliferation of human arterial SMCs in vitro (Hu et al, unpublished observations). Second, suramin has long been in clinical use, and no side effects in the mouse model have been observed. Third, it is technically easy to use for treatment of vein segments of bypass patients without prolonging surgery times. Finally, suramin is a smaller molecule that should easily penetrate human vessel walls. Therefore locally applied suramin might be effective for treatment of bypass patients.

At the early stage, the neointimal lesion has an inflammatory nature characterized by mononuclear cell infiltration of vein bypass grafts and followed by SMC proliferation.^{1 3} Activated monocytes and macrophages, which produce mitogenic, fibrogenic, and angiogenic factors that can influence tissue remodeling, are central to inflammation^{17 18} and may play a role in the development of neointimal hyperplasia in grafted veins. Suramin is used primarily for treatment of African trypanosomiasis by intravenous injection. The drug is slowly cleared up by the kidney, with a terminal elimination half-life of 50 days.¹⁹ In the current study, suramin mixed with pluronic-127 gel allows a slow release from the gel, although the time period and speed of local drug release remain to be quantified. Locally applied suramin might also influence the inflammatory process through interactions with the surface receptors of macrophages.

SMC accumulation expressing PDGF receptors is a hallmark of vein graft arteriosclerosis. What is the initial factor resulting in SMC proliferation in grafted veins? Surgical or traumatic and ischemic injury to the vein segments leading to inflammatory reactions may be partially responsible for SMC migration/proliferation at the early stage in the vein grafts. In addition, we believe that mechanical stress may play a crucial role in neointima formation by enhancing gene expression of growth factors, cytokines, and matrix proteins.²⁰ In grafted veins, mechanical force on the vessel segment suddenly increases >10-fold (arterial vs venous blood pressure), which provides a strong stimulus to vascular SMCs. We^{21 22} and others^{23 24} previously demonstrated that acutely elevated blood pressure, mechanical stress, or balloon injury to the carotid artery induces MAPK activation. Recently we observed that physical forces rapidly induced phosphorylation of PDGF receptor-, supporting the mechanical stress–stimulated activation of PDGF receptor-.⁹ Other reports have established mechanical stress–induced PDGF and fibroblast growth factor (FGF) production in SMCs.^{7 8 25 26} Interestingly, Mehta et al²⁷ demonstrated that external stenting onto the grafted vein reduced neointima lesions >70% through reduction of PDGF expression. Mechanical stresses may directly perturb the cell surface, alter receptor conformation, or stimulate PDGF and FGF production, thereby initiating signaling pathways used by growth factors. Thus suramin, irreversibly binding to growth factor receptors, prevents growth factor receptor activation and blocks MAPK-AP-1 signal transduction pathways, by which SMC migration and proliferation in vitro and in vivo are inhibited.

	Acknowledgments

 
This work was supported by grants P-13099-BIO and P12847-MED from the Austrian Science Fund and P6286 from the Jubiläumsfonds of the Austrian National Bank. Dr Hu is a recipient of an APART Stipend from the Austrian Academy of Sciences. We thank A. Jenewein and G. Sturm for excellent technical assistance.

Received January 27, 1999; revision received April 22, 1999; accepted April 22, 1999.

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