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(Hypertension. 2005;46:622.)
© 2005 American Heart Association, Inc.

Original Articles

Adhesion Molecule Expression in Fibroblasts

Alteration in Fibroblast Biology After Transfection With LOX-1 Plasmids

Kui Chen; Jiawei Chen; Yong Liu; Jin Xie; Dayuan Li; Tatsuya Sawamura; Paul L. Hermonat; Jawahar L. Mehta

From the Departments of Internal Medicine (K.C., J.C., J.L., J.X., D.L., T.S., P.L.H., J.L.M.) and Physiology and Biophysics (J.C., J.L.M.), University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Ark.

Correspondence to J.L. Mehta, MD, PhD, Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, 4301 W Markham St, Slot 532, Little Rock, AR 72205. E-mail MehtaJL{at}uams.edu

	Abstract

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The endothelial lectinlike, oxidatively (ox-) modified LDL receptor LOX-1 is a critical player in the pathogenesis of atherosclerosis and myocardial ischemia. Ox-LDL binding of LOX-1 results in the expression of various adhesion molecules, which attract monocytes to endothelial cells, an initial step in atherogenesis. We wished to examine the role of the ox-LDL/LOX-1 signaling pathway in fibroblasts, which naturally express low levels of LOX-1. Rat cardiac fibroblasts were transfected with either cytomegalovirus (CMV)-LOX-1^wt (amino acids [aa] 1 to 273) or CMV-LOX-1^1-261 (an ox-LDL–binding negative mutant, aa 1 to 261) plasmid. Western blots showed that LOX-1 protein expression was increased significantly in cells transfected with CMV-LOX-1^wt or CMV-LOX-1^1-261 plasmid (P<0.01 vs control). Fibroblasts transfected with CMV-LOX-1^wt showed ox-LDL binding, whereas fibroblasts without transfection and those transfected with CMV-LOX-1^1-261 did not bind ox-LDL. Compared with untransfected cells, ox-LDL treatment (50 µg/mL, 24 hours) markedly induced the expression of the leukocyte adhesion molecules intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM)-1 as well as matrix metalloproteinase (MMP)-1 in cells transfected with CMV-LOX-1^wt (P<0.05) but not in cells transfected with CMV-LOX-1^1-261. Concurrently, ox-LDL treatment enhanced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (P<0.05 vs control) in CMV-LOX-1^wt–transfected cells. These data suggest that in cardiac fibroblasts, ox-LDL binds to LOX-1 and activates p38 MAPK, followed by the expression of ICAM-1, VCAM-1, and MMP-1. Thus, fibroblasts transform into an endothelial phenotype on transfection with CMV-LOX-1^wt and subsequent exposure to ox-LDL. This study provides a useful model system (plasmid-transfected fibroblasts) to study the molecular biology of LOX-1.

Key Words: lipoproteins • fibroblasts • collagen • genetics

	Introduction

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Oxidatively modified low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis.^1,2 Ox-LDL influences the properties of various cells mainly by binding to cell surface receptors.^3,4 Several ox-LDL receptors, including scavenger receptor (SR)-A I/II, CD36, SR-BI, FcRII, lectinlike ox-LDL receptor-1 (LOX-1), and macrosialin, have been identified in macrophages, smooth muscle cells, endothelial cells, and platelets.^3,5–8 Among them, LOX-1 has received extensive attention because its expression is increased in several pathologic conditions, such as hypertension, diabetes, and atherosclerosis.^9–13

LOX-1 is mainly expressed in vascular endothelial cells and acts as a cell surface receptor for the binding, internalization, and degradation of ox-LDL.¹³ It is also an important target of therapeutic interventions because proatherogenic conditions such as diabetes and hypertension induce its expression.^12,14 In addition, the expression of monocyte adhesion molecules, which are closely associated with atherogenesis, is upregulated by ox-LDL/LOX-1 activation.^15,16

Fibroblasts produce collagens, which are involved in thickening of the intima of atherosclerotic lesions.^17,18 Fibroblasts also release matrix metalloproteinases (MMPs),¹⁹ which are relevant to plaque disruption and acute ischemic syndromes.²⁰ As such, fibroblasts are involved in cardiac remodeling by producing collagens as well MMPs.²¹ A balance between collagen synthesis and degradation determines plaque stability and vascular and myocardial stiffness. Our previous studies showed extensive expression of LOX-1 in atherosclerotic lesions²² as well as in the ischemic myocardium.²³ We have also shown that ox-LDL/LOX-1 activation stimulates MMP-1 and MMP-3 expression in endothelial cells.²⁴ However, the role of LOX-1 in fibroblasts in response to ox-LDL has not yet been studied.

In this study, we cloned the plasmids expressing wild-type LOX-1 (cytomegalovirus [CMV]-LOX-1^wt) or the ox-LDL–binding negative-mutant LOX-1, transfected them into rat cardiac fibroblasts, and examined the biologic response of these cells to ox-LDL treatment.

	Methods

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Construction of LOX-1 Expression Plasmids
Human LOX-1 cDNA was amplified by polymerase chain reaction (PCR). For wt LOX-1 (273 amino acid residues), the primers were 5'-CGT GAC TGC TTC ACT CTC TCA-3' (forward) and 5'-TTG GCA CCC AAG TGA CAA A-3' (reverse); for the ox-LDL–binding negative-mutant LOX-1 (261 amino acid residues, C-terminal deletion), the primers were 5'-CGT GAC TGC TTC ACT CTC TCA-3' (forward) and 5'-CTA GAA GGC AGC TAA AAT GCA G-3' (reverse). The cDNA was cloned into the pCMVTnTTM plasmid (Promega) with the use of EcoRI and XbaI restriction enzymes, and the DNA sequence was analyzed by the DNA Sequencing Laboratory at the University of Arkansas for Medical Sciences. No mutation was found. Hereafter, the plasmids expressing wt and mutant LOX-1 are referred to as CMV-LOX-1^wt and CMV-LOX-1^1-261, respectively.

Cell Culture and Transient Transfection
Rat cardiac fibroblasts were cultured as described earlier.²¹ The cells (fifth passage) were plated in 58-mm dishes (Nuncbrand) with 5 mL of culture medium. On reaching 70% confluence, cells were transfected with CMV-LOX-1^wt or CMV-LOX-1^1-261 with FuGENE 6 transfection reagent (Roche), according to the manufacturer’s suggestions. Five hours after transfection, the medium was replaced with fresh, serum-free medium, and the cells were starved for 48 hours before they were cultured in the complete culture medium with or without 50 µg/mL ox-LDL for 24 hours. Cells then were harvested and subjected to the studies described next.

Binding of Dil–Ox-LDL to Cardiac Fibroblasts
Forty-eight hours after transfection, the cells were incubated with 5 µg/mL 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled ox-LDL (Intracel Resources) in serum-free Dulbecco’s modified Eagle’s medium on ice for 1 hour and washed 5 times with phosphate-buffered saline. The fluorescence of DiI was observed with a fluorescence microscope.

RNA Extraction and Semiquantitative RT-PCR Analysis
After treatment, total cellular RNA was isolated from fibroblasts with the use of an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Equal amount of total RNA (3 µg) was reverse-transcribed (RT) with oligo(dT) and M-MLV reverse transcriptase (Promega) at 37°C for 1 hour. PCR amplification of rat procollagen type I, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and ß-actin was achieved with the use of specific primers. 5'-ACA GCA CGC TTG TGG AT-3' (forward) and 5'-GTC TTC AAG CAA GAG GAC CA-3' (reverse) were for procollagen type I; 5'-CTG TCG GTG CTC AGG TAT CC-3' (forward) and 5'-CCA ACT TCT CAG TCA CCT CC-3' (reverse) were for ICAM-1; 5'-CGC TCG CTC AGA TTG GAG AC-3' (forward) and 5'-CTC GCT GGC ACA TGT CAT CG-3' (reverse) were for VCAM-1; and 5'-GAG CTA TGA GCT GCC TGA CG-3' (forward) and 5'-AGC ACT TGC GGT CCA CGA TG-3' (reverse) were for ß-actin. PCR was performed in a volume of 50 µL containing 2 µL cDNA mixture, 2 mmol/L MgCl₂, 50 mmol/L KCl, 10 mmol/L Tris HCl (pH 8.3), 200 mmol/L dNTP, 1.25 U Taq polymerase, and 20 pmol/L primers. PCR was carried out with an initial 5-minute denaturation at 94°C, followed by 32 cycles of amplification (60 seconds at 94°C, 50 seconds at 60°C, 2 minutes at 72°C) and a final incubation of 10 minutes at 72°C. The PCR-amplified products were visualized in 1.5% agarose gels containing ethidium bromide. The relative intensity of all bands was normalized against the ß-actin bands, and the results were analyzed with UN-SCAN-IT gel software (Silk Scientific).

Western Blot Analysis
After treatment, fibroblasts were harvested and lysed in cell lysis buffer (Cell Signaling Technology). Lysates were centrifuged at 12 000 rpm for 20 minutes at 4°C, and the supernatants were collected. After protein concentration was measured with the Bio-Rad protein assay system, 50 µg protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and incubated with primary antibodies directed against procollagen type I (Santa Cruz), MMP-1 (Oncogene), ICAM-1 (Santa Cruz), VCAM-1 (Santa Cruz), LOX-1, and ß-actin (Sigma). Membranes were then washed and incubated with horseradish peroxidase–conjugated secondary antibody (Calbiochem), and the protein bands were detected with enhanced chemiluminescence reagents (Amersham).

Data Analysis
All experiments were repeated at least 3 times on different occasions. The data were expressed as mean±SD. Statistical significance was determined in multiple comparisons among groups of data in which ANOVA and Student’s t test indicated the presence of a significant difference. A value of P<0.05 was considered significant.

	Results

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Binding of DiI–ox-LDL and Expression of LOX-1 in Cardiac Fibroblasts
The expression of LOX-1 in CMV-LOX-1^wt– or CMV-LOX-1^1-261–transfected cardiac fibroblasts was determined by ox-LDL binding and Western blot analysis. We found that in the basal state, fibroblasts (control) showed negligible ox-LDL binding, which was increased (to 50% of total cells) in CMV-LOX-1^wt–transfected cells, whereas there was no ox-LDL binding in CMV-LOX-1^1-261–transfected cells (Figure 1, top). The transfection efficiency (50%) was suggested by transfection of fibroblasts with green fluorescent protein (GFP) expression plasmids and observation by fluorescence microscopy (Figure 1, top).

View larger version (43K): [in this window] [in a new window]   Figure 1. The binding activity of DiI–ox-LDL and the expression of LOX-1. Top, The binding of DiI–ox-LDL was undetected in untransfected control cells but was increased to 50% of total cells in CMV-LOX-1wt–transfected cells. It was consistent with the percentage of cells that express GFP after they were transfected with CMV-GFP plasmid. In contrast, binding activity was negligible in CMV-LOX-11-261–transfected cells. The upper panels are representative images captured during fluorescence microscopy, and the bottom panels are representative images from phase-contrast microscopy. Bottom, Western blotting showed that LOX-1 protein expression was low in control cells but was increased markedly in CMV-LOX-1wt– or CMV-LOX-11-261–transfected cells. Abbreviations are as defined in text.

LOX-1 protein expression under basal conditions or after ox-LDL treatment was relatively modest but increased significantly in CMV-LOX-1^wt– as well as in CMV-LOX-1^1-261–transfected fibroblasts (both P<0.01 vs control). The expression of LOX-1 in fibroblasts under basal conditions and after ox-LDL treatment was similar (Figure 1, bottom) and was much higher after CMV-LOX-1^wt or CMV-LOX-1^1-261 transfection. We believe that we successfully constructed the LOX-1 expression plasmids and transfected fibroblasts with these plasmids, and this process resulted in expression of LOX-1 in fibroblasts.

Expression of Procollagen Type I and MMP-1 in Cardiac Fibroblasts
To examine the effects of LOX-1 transfection on cardiac fibroblasts, we examined the expression of procollagen type I and MMP-1. The expression of procollagen type 1 (mRNA and protein) was similar in untransfected and both CMV-LOX-1^wt– and CMV-LOX-1^1-261–transfected cells and did not change after ox-LDL treatment (Figure 2, left). MMP-1 protein expression was also similar in the untransfected and CMV-LOX-1^wt– or CMV-LOX-1^1-261–transfected cells. However, after ox-LDL treatment, MMP-1 expression increased dramatically in CMV-LOX-1^wt–transfected cells (P<0.01 compared with untransfected cells), whereas it remained unchanged in CMV-LOX-1^1-261–transfected cells (Figure 2, right).

View larger version (25K): [in this window] [in a new window]   Figure 2. The expression of procollagen type I and MMP-1. Left, Western blot and RT-PCR showed that the expression of procollagen type I was similar in untransfected, CMV-LOX-1wt–transfected, or CMV-LOX-11-261–transfected cells and was not changed after ox-LDL treatment. Right, Western blotting showed that the expression of MMP-1 was similar in untransfected, CMV-LOX-1wt–transfected, or CMV-LOX-11-261–transfected cells under basal conditions. However, after ox-LDL treatment, MMP-1 expression was increased markedly in CMV-LOX-1wt–transfected cells, but it remained unchanged in CMV-LOX-11-261–transfected cells. Abbreviations are as defined in text.

Expression of ICAM-1 and VCAM-1 in Cardiac Fibroblasts
Under basal conditions, expression (mRNA and protein) of ICAM-1 and VCAM-1 was low and remained low after transfection of fibroblasts with CMV-LOX-1^wt or CMV-LOX-1^1-261 (Figure 3). This observation is consistent with the studies by Kacimi et al,²⁵ who showed minimal expression of ICAM-1 and VCAM-1 in cardiac fibroblasts. We wondered whether LOX-1 overexpression would alter the biology of these cells. We found that transfection with CMV-LOX-1^wt or CMV-LOX-1^1-261 had no effect on the expression of ICAM-1 or VCAM-1 in the basal state. However, after ox-LDL treatment, the expression of both ICAM-1 and VCAM-1 was significantly upregulated in the CMV-LOX-1^wt–transfected fibroblasts (P<0.05). The expression of both mRNA and protein was increased in CMV-LOX-1^wt–transfected fibroblasts. Note that there was no increase in ICAM-1 or VCAM-1 expression in fibroblasts transfected with CMV-LOX-1^1-261, even after the cells were treated with ox-LDL (Figure 3).

View larger version (46K): [in this window] [in a new window]   Figure 3. The expression of ICAM-1 and VCAM-1. Expression (mRNA and protein) of ICAM-1 and VCAM-1 was similar in untransfected (control), CMV-LOX-1wt–transfected, or CMV-LOX-11-261–transfected cells under basal conditions. However, after ox-LDL treatment, expression of ICAM-1 and VCAM-1 (both protein and mRNA) increased markedly in CMV-LOX-1wt–transfected cells, but it remained unaffected in CMV-LOX-11-261–transfected cells. Abbreviations are as defined in text.

p38 MAPK and p44/42 MAPK Activation in Cardiac Fibroblasts
Mitogen-activated protein kinase (MAPK) is an important intracellular signaling molecule and is activated during ox-LDL/LOX-1 activation in endothelial cells.¹⁵ We examined whether LOX-1 overexpression would affect MAPK activation in fibroblasts. We found that there was no difference in p38 MAPK expression and phosphorylation between transfected and untransfected cells. After ox-LDL treatment, p38 MAPK expression was also similar between transfected and untransfected cells. However, compared with that in untransfected cells, the phosphorylation of p38 MAPK was greatly stimulated in CMV-LOX-1^wt–transfected cells (P<0.05) but not in CMV-LOX-1^1-261–transfected cells (Figure 4). Note that the expression and phosphorylation of p44/42 MAPK remained unaffected by CMV-LOX-1^wt or CMV-LOX-1^1-261 transfection and were unaffected by ox-LDL treatment (Figure 4).

View larger version (53K): [in this window] [in a new window]   Figure 4. p38 and p44/42 MAPK activation. Western blotting showed that there was no difference in p38 and p44/42 MAPK expression among different groups of cells. However, after ox-LDL treatment, compared with that in untransfected cells, phosphorylation of p38 MAPK was greatly stimulated in CMV-LOX-1wt–transfected cells but not in CMV-LOX-11-261–transfected cells. In contrast, the phosphorylation of p44/42 MAPK remained unaffected by ox-LDL treatment in these cells.

	Discussion

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This study demonstrates a novel yet undefined alteration in the biology of cardiac fibroblasts, in that LOX-1^wt–transfected cells can express high levels of leukocyte adhesion molecules in response to ox-LDL. Furthermore, we have shown that fibroblasts can be used successfully to study LOX-1 mutants and their altered molecular biology. The key to this system is that fibroblasts express only a small amount of LOX-1 under basal conditions, and they can be adequately transfected with wt and mutant LOX-1 expression plasmids.

Transfection of Cardiac Fibroblasts With CMV-LOX-1^wt and CMV-LOX-1^1-261
Dr Sawamura’s group first cloned human LOX-1 cDNA and described that LOX-1 binds, internalizes, and degrades ox-LDL in COS-7 cells.¹³ We constructed a wt LOX-1 (aa 1 to 273) expression plasmid (CMV-LOX-1^wt) and an ox-LDL–binding negative-mutant LOX-1 (aa 1 to 261) expression plasmid (CMV-LOX-1^1-261). In this study, the wt and mutant LOX-1 expression plasmids were successfully transfected into rat cardiac fibroblasts. It was confirmed by the finding that both CMV-LOX-1^wt– and CMV-LOX-1^1-261–transfected fibroblasts expressed a large amount of LOX-1, whereas untransfected fibroblasts expressed only low levels of LOX-1 (Figure 1, bottom). Furthermore, after transfection, the percentage of cells (CMV-LOX-1^wt transfected) that bound ox-LDL was similar to that expressing GFP (CMV-GFP transfected), which served as a transfection efficiency marker (Figure 1, top).

This study has also demonstrated the requirement of the last 12 aa residues at the C-terminus of LOX-1 for ox-LDL recognition and binding, in keeping with the results of an earlier study.²⁶ We confirmed this phenomenon by showing that only cells overexpressing wt LOX-1 (aa 1 to 273), but not the mutant LOX-1 (aa 1 to 261), could bind DiI–ox-LDL. After ox-LDL treatment, expression of MMP-1, ICAM-1, and VCAM-1 was significantly increased in fibroblasts transfected with CMV-LOX-1^wt but not in those transfected with CMV-LOX-1^1-261. In addition, we found that ox-LDL treatment increased the phosphorylation of p38 MAPK, but not of p44/42 MAPK, in fibroblasts transfected with CMV-LOX-1^wt but not with CMV-LOX-1^1-261. Taken together, these data suggest that the binding of ox-LDL to LOX-1 is required for its effects to manifest in fibroblasts.

Biologic Function of LOX-1 in Cardiac Fibroblasts
Cardiac fibroblasts are the main source of collagen type I, which accumulates in the heart and leads to fibrosis and heart failure.^21,27,28 Cardiac fibroblasts also release MMP-1 that degrades collagen type I. Under physiologic conditions, there is a balance between collagen type I production and degradation. However, this balance could be disrupted by several pathologic conditions. For example, anoxia/reoxygenation stimulates the expression of both collagen type I and MMP-1,²⁹ whereas angiotensin II enhances the expression of collagen type I but inhibits the expression of MMP-1.²¹ In the present study, we examined whether ox-LDL/LOX-1 activation would influence the expression of collagen type I and MMP-1 in cardiac fibroblasts and found that ox-LDL had no effect on procollagen type I expression in untransfected, CMV-LOX-1^wt–transfected, or CMV-LOX-1^1-261–transfected fibroblasts. However, LOX-1 upregulation (via plasmid transfection), together with ox-LDL treatment, increased MMP-1 expression markedly. It has been suggested that the release of MMPs is a major factor by which the fibrous cap in atherosclerotic regions softens and finally dissolves, leading to platelet accumulation.^30,31 Ox-LDL accumulation and LOX-1 upregulation have been found in rupture-prone atherosclerotic plaques.^22,32 We showed earlier that ox-LDL–treated endothelial cells release large amounts of MMP-1 and MMP-3 and that this effect of ox-LDL can be blocked by pretreatment of the cells with LOX-1 antibody, suggesting the role of LOX-1.²⁴ In this article, we have shown that ox-LDL can also increase MMP-1 expression in fibroblasts via LOX-1.

Normally, fibroblasts express only low levels of the leukocyte adhesion molecules ICAM-1 and VCAM-1, even after ox-LDL treatment. In this study, we showed that fibroblasts transfected with CMV-LOX-1^wt, but not CMV-LOX-1^1-261, expressed high levels of ICAM-1 and VCAM-1, both mRNA and protein, in response to ox-LDL. This alteration in the biology of fibroblasts gives them the functional characteristics of endothelial cells.

Our observations gain support from a study by Yoshikawa et al,³³ who showed that tumor necrosis factor-– and interleukin-4–stimulated fibroblasts facilitate the accumulation of monocytes at inflammation sites. In our preliminary studies in high-cholesterol-diet–fed, LDL-receptor–knockout mice, we found well-developed atherosclerosis and intense expression of CD68 throughout the thickness of the aorta, implying that monocyte/macrophage adhesion is present not just in the subintima but also in the adventitial region, where fibroblasts are present. All of these regions also express high levels of LOX-1 (authors’ unpublished data).

MAPK is considered a common intracellular signaling molecule that responds to extracellular stimuli.^34,35 We examined the protein expression and phosphorylation of both p38 and p44/42 isoforms and found that p38 MAPK is involved in the ox-LDL–induced upregulation of MMP-1, ICAM-1, and VCAM-1 in CMV-LOX-1^wt–transfected fibroblasts. In previous studies,^21,36,37 we showed that ox-LDL increases the expression of adhesion molecules in endothelial cells via p38 MAPK and nuclear factor-B activation. Furthermore, inhibition of p38 MAPK abolished these ox-LDL–induced effects. Here we have demonstrated a potentially important role for p38 MAPK activation in a series of reactions as a result of ox-LDL binding to LOX-1 in fibroblasts.

In summary, we have shown in the present study that both CMV-LOX-1^wt and CMV- LOX-1^1-261 plasmids can be successfully transfected into cardiac fibroblasts. The transfection of CMV-LOX-1^wt results in a functional alteration and transduction pathway activation in cardiac fibroblasts similar to those in endothelial cells in response to ox-LDL. Although LOX-1 expression increased in cells transfected with either CMV-LOX-1^wt or CMV-LOX-1^1-261, only CMV-LOX-1^wt transfection resulted in the binding of DiI–ox-LDL. Furthermore, ox-LDL treatment only increased the expression of MMP-1, ICAM-1, and VCAM-1 and the activation of p38 MAPK in CMV-LOX-1^wt–transfected cells.

Perspective
Understanding fibroblast function in vascular biology is gaining renewed attention. These cells play a potent role in atherosclerosis, in both plaque rupture and vascular remodeling, including the formation of aneurysms. These cells also have an important role in cardiac remodeling as a result of ischemic and nonischemic cardiomyopathy. All of these conditions are associated with a marked increase in oxidative stress. The alterations in fibroblast function described herein provide a novel link between oxidative stress, ox-LDL, and LOX-1 in the biology of fibroblasts. Furthermore, these studies show that fibroblasts are a valuable system for studying the molecular biology and function LOX-1.

	Acknowledgments

 
This work was supported by a Scientist Development Grant from the American Heart Association, a merit review grant from the Department of Veteran Affairs Merit Review, and a grant from the National Institutes of Health.

Received March 2, 2005; first decision March 14, 2005; accepted July 13, 2005.

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