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(Circulation. 2002;105:1220.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Tumor Necrosis Factor- Regulates Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor Binding Protein-3 Expression in Vascular Smooth Muscle

A. Anwar, MD; A.A. Zahid, MD; K.J. Scheidegger, PhD; M. Brink, PhD; P. Delafontaine, MD

From the Division of Cardiology, University Hospital of Geneva, Switzerland and Division of Cardiovascular Diseases (P.D.), Kansas University Medical Center, Kansas City, Kan.

Correspondence to P. Delafontaine, MD, FACC, Division of Cardiovascular Diseases, Kansas University Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7378. E-mail pdelafontaine{at}kumc.edu

	Abstract

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Background— Inflammatory mediators such as tumor necrosis factor- (TNF-), interleukin 1ß (IL-1ß), IL-6, and interferon (IFN-) may change coronary plaque integrity by altering vascular smooth muscle cell (VSMC) survival and modifying the extracellular matrix. Insulin-like growth factor-1 (IGF-1) prevents apoptosis, promotes matrix formation, and can decrease TNF- or IL-1ß–induced proteoglycan degradation.

Methods and Results— To determine the effects of cytokines on the IGF-1 system, rat aortic VSMCs were exposed to TNF- (10 to 500 ng/mL), IL-1ß (20 pg to 10 ng/mL), IL-6 (100 pg to 15 ng/mL), or IFN- (10 to 600 U/mL). IL-1ß, IL-6, and IFN- did not regulate IGF-1, IGF-1 receptor (R), or IGF binding proteins (IGFBPs). However, TNF- markedly decreased IGF-1 mRNA (85% reduction at 24 hours) and increased IGFBP-3 mRNA and protein (300% increase at 24 hours). These changes were blocked by actinomycin D, consistent with a transcriptional mechanism. Experiments using TNF binding protein-1 indicated that these effects were not attributable to secretion of an autocrine factor. Anti–IGFBP-3 antibodies increased VSMC DNA synthesis 3-fold. In addition, apoptosis induced by TNF-, IFN-, and Fas ligand was markedly reduced by desamino-(1-3)-IGF-1.

Conclusions— TNF-, a cytokine that is upregulated in atherosclerotic plaques, reduces IGF-1 and increases IGFBP-3 in VSMCs, likely leading to a reduction in bioactive IGF-1. Because IGF-1 is important for growth and survival of VSMCs, its downregulation by TNF- possibly plays a crucial role in acute and chronic coronary syndromes by decreasing VSMC viability and promoting plaque instability.

Key Words: cells • growth substances • muscle, smooth • apoptosis • atherosclerosis

	Introduction

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Insulin-like growth factor-I (IGF-1), acting via its tyrosine kinase receptor (IGF-1R), regulates normal developmental growth through endocrine and autocrine/paracrine mechanisms.¹ IGF-1 may also play a role in mechanisms of cellular growth and survival in cardiovascular tissues in pathologic states.²

In atherosclerotic lesions, inflammatory processes change the structural integrity of the coronary plaque,³ and activated macrophages are known to produce cytokines, proteolytic enzymes, and growth factors that alter growth and survival of vascular smooth muscle cells (VSMCs) and modify the extracellular matrix.⁴ Tumor necrosis factor- (TNF-), interleukin 1ß (IL-1ß), interleukin 6 (IL-6), and interferon (IFN-) are implicated in this process. IGF-1 promotes matrix formation and can decrease cytokine-induced proteoglycan degradation.^5,6 Furthermore, IGF-1 prevents apoptosis,⁷ and its activity is modulated by IGF-1 binding proteins (IGFBPs), including IGFBP-3.¹

IGFBP-3, the most abundant carrier protein for IGF-1, may also function as a negative regulator of cell growth and as a proapoptotic factor. Thus, transfection of IGFBP-3 into IGF-1R–negative fibroblasts inhibits the proliferative response to serum,⁸ and exogenous IGFBP-3 inhibits growth of Hs578T human breast cancer cells.⁹

To obtain insight into potential effects of TNF-, IL-1ß, IL-6, and IFN- on the vascular IGF-1 system, we assessed effects of these cytokines on VSMC IGF-1, IGF-1R, and IGFBP expression. Our findings have important implications for understanding mechanisms controlling VSMC growth and viability in vivo, particularly in atherosclerotic plaques.

	Methods

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Cell Culture
Rat aortic VSMCs isolated by enzymatic digestion¹⁰ were quiesced in DMEM/Ham F-12 for 48 hours before treatment with 10 to 500 ng/mL TNF-, 10 to 600 U/mL mouse recombinant IFN-, 20 pg to 10 ng/mL rat recombinant IL-1ß, or 100 pg to 15 ng/mL rat recombinant IL-6 for 0 to 24 hours. For some experiments, quiescent cells were coincubated in the presence or absence of 50 ng/mL TNF- with or without actinomycin D (10 µg/mL, 24 hours) or cycloheximide (10 µg/mL, 24 hours). For other experiments, conditioned medium from cells treated with or without 50 ng/mL TNF- for 12 hours was coincubated with or without 25 µg/mL of TNF binding protein-1 (TNFBP-1) for 30 minutes and then added to a new set of cells for 24 hours. Total RNA and conditioned media were harvested.

Northern Analysis
Samples of 20 µg total RNA were size fractioned by agarose-formaldehyde gel electrophoresis and transferred, and membranes were prehybridized and then hybridized for 1 hour at 68°C in a QuickHyb solution (Stratagene) with denatured herring sperm DNA and 1.25x10⁶ cpm/mL of ³²P-labeled IGFBP-3 and GAPDH cDNA probes before washing and autoradiography. Densitometric data were normalized for the GAPDH signal.

RNase Protection Assays
RNase protection assays were performed as previously described.¹⁰ In brief, 20-µg samples of total RNA were hybridized with [³²P]UTP-labeled antisense IGF-1 and IGF-1R riboprobes and cohybridized with a GAPDH riboprobe before analysis by 6% polyacrylamide 8 mol/L urea denaturing gel electrophoresis. Densitometry was performed by PhosphorImager.

Western Ligand Blotting
Conditioned media were acidified with 2N acetic acid, concentrated, lyophilized, and resuspended in sodium phosphate buffer. Western ligand blotting was performed according to the method of Hossenlop et al¹¹ with 125I-IGF-1. Rinsed membranes were dried and exposed to film, and band intensity was quantified by densitometry.

TUNEL Assay
VSMCs were incubated for 48 hours with various combinations of cytokines (200 ng/mL TNF-, 400U/mL IFN-, and 100 ng/mL Fas ligand) alone or with 100 ng/mL desamino-(1-3)-IGF-1 (des-IGF-1). The cells were washed with PBS, fixed with 4% paraformaldehyde, rehydrated, exposed to 0.3% H₂O₂ in methanol for 20 minutes, permeabilized with 0.1% Triton X-100, and rinsed, and the TUNEL assay was performed according to the manufacturer’s instructions (in situ cell death detection, POD Kit, Roche Diagnostic). After washing, slides were incubated for 5 minutes with 1 µg/mL fluorescent DNA probe 4,6-diamindino-2-phenylindole. Slides were analyzed under a Zeiss fluorescence microscope. Staurosporine (1 µmol/L) was used as a positive control. Ten fields were counted (100 cells per field) by 2 independent observers. The experiment was repeated 3 times.

Caspase-3 Assay
Apoptosis was also quantified by measuring activity of caspase-3 (an important mediator of programmed cell death) using the ApoAlert caspase colorimetric assay kit (Clontech). Briefly, VSMCs were incubated for 48 hours with cytokines (TNF- 200 ng/mL, IFN- 400 U/mL, and Fas ligand 100 ng/mL) alone or with 100 ng/mL des-IGF-1. Cells were subsequently washed with PBS, trypsinized, and centrifuged at 400g for 10 minutes and resuspended in 50 µL of chilled cell lysis buffer and incubated on ice. Five milliliters of 1 mmol/L caspase-3 substrate (DEVD-pNitroaniline) and 50 µL of 2xreaction buffer/DTT mix were added, and the samples were incubated at 37°C for 1 hour. Protease activity was quantified by spectrophotometric changes at 405 nm. To confirm the correlation between protease activity and signal detection, a control reaction was set up by incubating lysates from TNF-–, IFN-–, and Fas ligand–treated cells with specific caspase-3 inhibitor (DEVD-CHO) before exposure to substrate to verify inhibition of caspase activity.

[³H]Thymidine Incorporation
Quiescent VSMCs in 24-well plates were incubated for 24 hours with 1 µCi/mL [³H]thymidine in serum-free medium alone or with 1:100 dilution of normal rabbit serum or anti–IGFBP-3 polyclonal antibody with or without TNF- 50 ng/mL and incorporation of [³H]thymidine measured as previously described.¹⁰ All experiments were performed in quadruplicates.

Statistics
Data are presented as mean±SEM. Statistical analysis was performed using Student’s t test or ANOVA when appropriate to analyze differences between groups. Significance was established when P<0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of TNF- on IGF-1 and IGF-1R
Exposure to TNF- (50 ng/mL) caused a rapid reduction in IGF-1 mRNA levels, detectable at 3 hours and reaching 85% decrease at 24 hours (n=6, not shown). Exposure of VSMCs to increasing doses of TNF- for 24 hours indicated a near-maximal reduction with 50 ng/mL (Figures 1A and 1B, n=5, P<0.001). In contrast, TNF- caused a small dose-dependent increase in IGF-1R mRNA levels, which became statistically significant at 50 ng/mL (Figures 1A and 1C, n=4, P<0.001).

View larger version (49K): [in this window] [in a new window]   Figure 1. Effect of TNF- on IGF-1 and IGF-1R. A, Representative RNase protection assay for IGF-1 and IGF-1R. Total RNA (20 µg/lane) was cohybridized to 32P-labeled IGF-1, IGF-1R, and GAPDH antisense riboprobes. B and C, Quantitative data from RNase protection assays showing effect of TNF- on IGF-1 (B) and IGF-1R (C) mRNA levels (mean±SEM, n=5).

To determine mechanisms, we incubated cells with or without TNF- in the presence or absence of actinomycin D. Actinomycin D alone did not change IGF-1 mRNA levels at 24 hours, consistent with the long half-life of this mRNA, but completely inhibited the ability of TNF- to reduce IGF-1 mRNA at 24 hours. It should be noted that actinomycin D reduced IGF-1R mRNA and GAPDH mRNA levels at 24 hours (Figure 2A). These findings indicated that TNF- downregulation of IGF-1 was transcriptionally mediated. Furthermore, inhibition of protein synthesis with cycloheximide almost completely blocked TNF-–induced downregulation of IGF-1 (Figure 2B).

View larger version (83K): [in this window] [in a new window]   Figure 2. Effects of Actinomycin D and Cycloheximide on TNF-–induced IGF-1 and IGF-1R mRNA expression. A, Representative RNase protection assays of VSMCs incubated alone (C) or with actinomycin D (Act.D), TNF-, or Act.D and TNF- for 24 hours. B, Representative RNase protection assays of VSMCs incubated alone (C) or with TNF-, cycloheximide (Cycl.), or TNF- and Cycl. for 24 hours.

Effect of TNF- on IGFBP-3
As assessed by Western ligand blotting, rat aortic VSMCs secrete primarily IGFBP-4 and IGFBP-2 and trace amounts of IGFBP-3. TNF- caused a marked increase in IGFBP-3 secretion with no change in other IGFBPs (Figure 3A). The increase in IGFBP-3 secretion with 50 to 100 ng/mL TNF- was already detectable at 6 hours (data not shown). Northern analysis showed a marked induction of IGFBP-3 mRNA expression in cells treated with increasing doses of TNF- for 24 hours (Figure 3B). In preliminary experiments, we found that TNF- induction of IGFBP-3 mRNA expression was already detectable at 3 hours and increased progressively over 24 hours.

View larger version (80K): [in this window] [in a new window]   Figure 3. Effects of TNF- on IGFBP-3 secretion. A and B, VSMCs were exposed to increasing doses of TNF- for 24 hours, and Western ligand blot of conditioned medium (A) or Northern blot analysis (B) was performed. C, Western ligand blot analysis of conditioned medium from cells exposed alone (C) or with actinomycin D (Act.D), TNF-, or TNF- and Act.D for 24 hours. D, Northern blot analysis of RNA from VSMCs incubated with or without (C) TNF- in the presence or absence of cycloheximide (Cycl.).

Inhibition of RNA synthesis using actinomycin D completely blocked the induction of IGFBP-3 mRNA expression by TNF- (not shown) and IGFBP-3 protein secretion (Figure 3C). Furthermore, cycloheximide blocked TNF- induction of IGFBP-3 mRNA expression (Figure 3D) and IGFBP-3 protein secretion (not shown).

Effect of TNFBP-1 on IGF-1 and IGFBPs
To determine whether an autocrine factor produced in response to TNF- was involved in the downregulation of IGF-1 and the induction of IGFBP-3, we incubated VSMCs for 24 hours in conditioned medium harvested from cells exposed to TNF- for 12 hours. The expected decrease in IGF-1 expression occurred but was completely blocked when the conditioned medium was preincubated in the presence of TNFBP-1 (Figure 4). TNFBP-1 also completely inhibited the ability of the conditioned medium to induce IGFBP-3 expression (not shown). These results indicate that the effects of TNF- on IGF-1 and IGFBP-3 were not mediated by secretion of an autocrine factor.

View larger version (88K): [in this window] [in a new window]   Figure 4. Effect of TNFBP-1 on TNF-–induced downregulation of IGF-1 and IGF-1R expression. VSMCs were exposed with or without TNF- (50 ng/mL) for 12 hours, and the conditioned medium was harvested and added to fresh VSMCs for 24 hours in the presence or absence of TNFBP-1 (25 ng/mL). Shown is a representative RNase protection assay. Preincubation of conditioned medium from TNF-–treated cells with TNFBP-1 completely inhibited its ability to downregulate IGF-1.

Effect of IFN-, IL-6, and IL-Iß on IGF-1, IGF-1R, and IGFBPs
IFN- (0 to 600 U/mL, 24 hours), IL-6 (0 to 15 ng/mL, 24 hours), or IL-1ß (0 to 10 ng/mL, 24 hours) did not regulate IGF-1 mRNA (IFN-, 102±2% control; IL-6, 96±12% control; and IL-Iß, 102±12% control; mean±SEM, n=3, P=NS) nor IGF-1R mRNA expression (IFN-, 106±8% control; IL-6, 102±5% control; and IL-1ß, 101±2% control; mean±SEM, n=3, P=NS). Additionally, these cytokines did not alter IGFBP production by VSMCs.

Des-IGF-1 Inhibits TNF-, IFN-, and Fas Ligand–Induced Caspase-3 Activity and Apoptosis
TNF- alone (200 ng/mL, 48 hours) did not induce apoptosis of VSMCs, consistent with the known resistance to apoptosis in these cells.¹² Therefore, 2 combinations of cytokines were used, TNF-/IFN- and TNF-/IFN-/Fas ligand. As shown in Figure 5 (representative picture) and Figure 6 (quantitative data), both combinations produced significant rates of apoptosis as determined by TUNEL assays. Des-IGF-1 inhibited TNF-/IFN- and TNF-/IFN-/Fas ligand–induced apoptosis by 66% and 73%, respectively. To confirm these findings, we measured activity of caspase-3, an enzyme that is critically important in the execution phase of apoptosis. Caspase-3 activity was determined basally and after exposure to TNF-/IFN-/Fas-ligand with or without 100 ng/mL des-IGF-1. As shown in Figure 7, TNF-/IFN-/Fas ligand markedly induced caspase-3 activity in VSMCs, and this induction was inhibited by des-IGF-1 by 55% (P<0.001). Des-IGF-1 instead of IGF-1 was used in these experiments to avoid interaction with IGFBPs, because des-IGF-1 is 100-fold less effective than IGF-1 for its ability to bind to IGFBPs.¹³

View larger version (30K): [in this window] [in a new window]   Figure 5. In situ 3' end labeling of DNA fragments (TUNEL). Representative photomicrographs of VSMCs demonstrating apoptosis as assessed by TUNEL staining of control (B) and cytokine-treated (D) cells. The same cells were also stained with 4,6-diamindino-2-phenylindole (DAPI) (control, A; cytokine-treated cells, C). Cytokines were TNF- 200 ng/mL, IFN- 400 U/mL, and Fas ligand 100 ng/mL.

View larger version (15K): [in this window] [in a new window]   Figure 6. Des-IGF-1 inhibits VSMC apoptosis induced by TNF-, IFN-, and Fas ligand. Effect of TNF- and IFN- or TNF-, IFN-, and Fas ligand with or without 100 ng/mL des-IGF-1 on apoptotic index of VSMCs. TUNEL staining was performed as described in the Methods section. Shown is the mean±SEM, n=3.

View larger version (16K): [in this window] [in a new window]   Figure 7. Des-IGF-1 inhibits TNF-–, IFN-–, and Fas ligand–induced caspase-3 activity in VSMCs. VSMCs were incubated in the presence of TNF- 200 ng/mL, IFN- 400 U/mL, and Fas ligand 100 ng/mL (cytokines) with or without 100 ng/mL des-IGF-1 for 48 hours, and caspase-3 activity was measured in cell lysates as described in the Methods section. Control indicates results using lysates from cytokine-treated cells incubated with a specific caspase-3 inhibitor (DEVD-CHO). Shown is the mean±SEM, n=3.

Effect of TNF- on [³H]Thymidine Incorporation
TNF- caused a small, non–statistically significant decrease in [³H]thymidine incorporation (Figure 8). Anti-IGFBP-3 antiserum markedly increased [³H]thymidine incorporation compared with normal rabbit serum (P<0.001), consistent with a growth-inhibitory effect of this binding protein. TNF- did not significantly alter DNA synthesis in the presence of anti-IGFBP-3 antibody or normal rabbit serum.

View larger version (19K): [in this window] [in a new window]   Figure 8. Effects of anti-IGFBP-3 antibody on [3H]thymidine incorporation. Quiescent VSMCs were treated with normal rabbit serum (NRS) or anti–IGFBP-3 antibody (-BP3) at 1:100 dilution with or without TNF- 50 ng/mL for 24 hours. Thymidine incorporation was measured as described in the Methods section; mean±SEM, n=5.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our data indicates that TNF- markedly suppresses IGF-1 mRNA expression in cultured VSMCs. The effect is dose and time dependent and is inhibited by actinomycin D, suggesting that synthesis of an intermediate signaling molecule or transacting factor is required. Concurrently, TNF- dramatically upregulates IGFBP-3 mRNA levels and secretion of IGFBP-3 protein from VSMCs, likely also through a transcriptional mechanism. Both effects require protein synthesis, because they are blocked by cycloheximide. TNF-, on the other hand, slightly upregulates IGF-1R mRNA expression in VSMCs.

To our knowledge, this is the first report showing that TNF- regulates IGF-1 in the cardiovascular system. In addition to regulating VSMC growth and migration,³ IGF-1 is a potent survival factor for many tissues, including VSMC and cardiac myocytes.¹⁴ It has previously been shown that TNF- has either no effect on IGF-1 expression in Sertoli cells¹⁵ or decreases IGF-1 expression in rat Leydig cells¹⁶ and in mouse osteoblasts.¹⁷ In macrophages, TNF- increases IGF-1 expression¹⁸ and IGF-1 stimulates monocyte and macrophage TNF- production.¹⁹ In rat hepatocytes, TNF- does not affect basal IGF-1 synthesis but blunts growth hormone induction of IGF-1.²⁰

TNF- is a proinflammatory cytokine with pleiotropic cellular effects and at high concentrations may induce cachexia, microvascular coagulation, and hemodynamic collapse.^21,22 TNF- is expressed by VSMCs in atheromatous plaques,²³ regulates their migration,²⁴ and may have no effect on VSMC growth¹² or increase VSMC proliferation 3-fold.²⁵ TNF- alone²⁶ or in combination with other cytokines¹² stimulates apoptosis of VSMCs. Furthermore, TNF- activates monocytes, endothelial cells, and macrophages. In contrast to these proatherogenic functions, TNF- may have antiatherogenic functions through inhibition of lipoprotein lipase and decreasing scavenger receptor activity.²⁷

The ability of TNF- to downregulate VSMC IGF-1 and upregulate IGFBP-3 is likely a direct effect. Thus, although TNF- has been shown to regulate cytokine production by VSMCs, neither IFN-, IL-1ß, nor IL-6 regulates VSMC IGF-1 or IGFBP-3 synthesis. To determine whether an autocrine factor produced in response to TNF- was involved, we incubated VSMCs for 24 hours in conditioned medium from cells exposed to TNF- for 12 hours. The expected decrease in IGF-1 and increase in IGFBP-3 expression occurred and was completely blocked when the conditioned medium was preincubated in the presence of TNFBP-1. This strongly suggests that the effect was mediated directly by TNF- and not by an additional autocrine factor.

TNF- increases IGFBP-3 in human chondrocytes²⁸ and in porcine Sertoli cells.¹⁵ IGFBP-3 may have stimulatory or inhibitory effects on IGF-1 action depending on the cell system. Our data demonstrating increased DNA synthesis in response to anti–IGFBP-3 antibody are consistent with an antiproliferative action of IGFBP-3 on VSMCs. Inhibitory effects of IGFBP-3 may result from sequestration of extracellular IGF-1 and possibly from an IGF-1 independent mechanism. Thus, IGFBP-3 predisposes cells to programmed cell death,²⁹ and its expression is upregulated by proapoptotic stimuli, including p53.³⁰ Furthermore, the antiproliferative effect of TNF- on human salivary gland tumor cells³¹ and MCF-7 breast cancer cells³² is mediated at least in part via IGFBP-3. Valentinis et al⁸ reported growth inhibition by overexpressing IGFBP-3 in fibroblasts that did not express IGF-1R. Recently, lower circulating IGFBP-3 levels have been associated with severity of coronary arteriosclerosis in men.³³

Our findings have far-reaching implications for understanding the control of VSMC growth and viability in vivo. Although there is increasing evidence for a role of inflammation in atherogenesis, the relation between TNF- and VSMC growth and survival remains obscure. It is likely that subpopulations of VSMCs in the atheromatous plaque may respond differently to cytokines and growth factors. The variable effects of TNF- on VSMC growth in vitro suggest that the effects of TNF- in vivo could be variable and depend, in part, on the balance between the effects of other cytokines and growth factors and on VSMC phenotype. The dramatic downregulation of IGF-1 and upregulation of IGFBP-3 induced by TNF- is a potentially powerful antiproliferative and proapoptotic stimulus for VSMCs. In our cultured cells, TNF- alone did not induce a significant change in [³H]thymidine incorporation, which is consistent with the report of Geng et al.¹² However, although cultured rat aortic VSMCs are particularly robust and resistant to proapoptotic stimuli, we were able to promote apoptosis of these cells with the combination of TNF- and IFN- with or without Fas ligand, and this effect was inhibited by des-IGF-1. We chose des-IGF-1 to avoid interference with IGF binding proteins.¹³ Our findings are consistent with the report that IGF-1 inhibited TNF-–induced cell death in 3T3 fibroblasts⁷ and colon cancer cells.³⁴

Our data have been obtained in a cell culture system using rodent VSMCs, and it will be important to verify these observations by studying human tissues. However, because TNF- is expressed at high levels in atherosclerotic plaques,²³ one can hypothesize that TNF-–induced downregulation of IGF-1 and upregulation of IGFBP-3 contribute to VSMC loss and plaque instability. Indeed, plaques prone to erosion and rupture have increased inflammatory cells and a relative reduction in VSMC number.^3,4 By analogy, we have recently shown that oxidized LDL expression in human atherosclerotic plaques is associated with VSMC apoptosis³⁵ and that oxidized LDL downregulates VSMC IGF-1 and IGF-1 receptor expression in vitro.¹⁰

In summary, we show that the cytokine TNF- potently inhibits IGF-1 and increases IGFBP-3 expression in VSMCs. This differential regulation of IGF-1 and IGFBP-3 likely plays an important role in the proapoptotic effect of TNF- on VSMCs. These findings have major implications for understanding the control of VSMC growth and viability in pathologic states and particularly in atherosclerotic plaque.

	Acknowledgments

 
This work is supported by National Institutes of Health grants HL47035, HL45317, and DK45215, and by the Swiss National Research Foundation grant FNSR3100-050799.97, the Fonds Gerbex-Bourget, and the Swiss Cardiology Foundation. We thank Laura Deaver for editorial assistance and Dr J. Pugin, Geneva University Hospital, for providing recombinant TNFBP-1.

Received November 8, 2001; revision received December 28, 2001; accepted January 2, 2002.

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