Big Mitogen-Activated Protein Kinase (BMK1)/ERK5 Protects Endothelial Cells From Apoptosis
Blood flow that is steady and laminar is known to be atheroprotective. One likely mechanism is enhanced endothelial cell (EC) survival. Because the mitogen-activated protein kinases (MAPKs) are known regulators of cell survival, we investigated the role of Big MAPK-1 (BMK1 or ERK5), which is potently stimulated by fluid shear stress. To activate BMK1, we overexpressed constitutively active (CA)-MEK5 in bovine lung microvascular ECs (BLMECs). Cell apoptosis was induced by growth factor deprivation (0% serum for 24 hours). Analysis of cell viability with MTT assay showed that activation of BMK1 by CA-MEK5 significantly improved cell viability from 48% to 87% and decreased apoptotic cells from 49% to 10%. Growth factor deprivation induced caspase-3 activity 5.2-fold, which was inhibited (≈60%) by CA-MEK5 overexpression. In contrast, inhibiting BMK1 activity by overexpressing dominant-negative BMK1 (DN-BMK1) stimulated apoptosis in BLMECs. Steady laminar fluid shear stress inhibited BLMEC apoptosis, and this protective effect was also reduced significantly by overexpressing DN-BMK1. Analysis of antiapoptotic mechanisms showed that both shear stress and CA-MEK5 stimulated phosphorylation of Bad on Ser112 and Ser136, whereas DN-BMK1 inhibited phosphorylation. Phosphorylation of Bad induced by BMK1 activation was independent of Akt, PKA, or p90RSK kinase activity. These results suggest that BMK1 activation by steady laminar flow is atheroprotective by inhibiting EC apoptosis via phosphorylation of Bad.
Endothelial cell (EC) integrity is important for maintenance of vessel function. EC apoptosis may contribute to atherosclerosis by increasing permeability of the endothelial monolayer and uptake of lipids in the vessel wall.1 EC apoptosis also contributes to plaque destabilization and thrombosis as suggested by increased EC apoptosis and circulating EC-derived microparticles in patients with unstable atherosclerotic plaques.2 Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) appear to be important stimuli for EC apoptosis and induce expression of EC genes that may promote atherosclerosis.
Blood flow with physiological levels of steady laminar shear stress has been shown to exert potent antiapoptotic and antiatherosclerotic effects.3 In contrast, flow that has low mean shear stress and turbulence is strongly correlated with EC dysfunction, EC apoptosis and atherosclerosis.4,5 In vitro studies showed that lack of shear stress triggers apoptosis in vascular ECs.6,7 However, steady laminar flow decreases EC apoptosis and blocks TNF-mediated JNK activation.7,8
Steady laminar shear stress has been proposed to inhibit EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (eg, superoxide dismutase), by inhibiting ASK1, and by stimulating nitric oxide-dependent pathways via PI3-kinase and Akt.9–12 Our laboratory has found that physiological shear stress inhibits TNF activation of JNK in ECs,8,11 and big mitogen-activated protein kinase 1 (BMK1) appears to play an important role in shear stress–mediated regulation of TNF signaling.13
The phenotype of BMK1-null mice strongly suggests that BMK1 is important for maintenance of vessel function. Specifically, BMK1-null mice display defective EC morphology, blood vessel formation, and cardiac development leading to embryonic lethality.14,15 In contrast, ERK1 and ERK2 knockout mice have no cardiovascular phenotype. BMK1 has been shown to phosphorylate transcription factors including c-Myc, NF-κB, Ets class transcription factor Sap1a, and myocyte enhancer–binding factor 2C (MEF2C), suggesting multiple cellular effects.16–19
Several recent studies show that BMK1 is important for cell proliferation and survival. BMK1 contributes to EGF-stimulated cell proliferation, Cot-induced NIH3T3 cell transformation17,20 and PC12 cell survival.21 Because of the strong BMK1 activation by flow and inhibition of EC apoptosis by flow, we investigated the antiapoptotic function of BMK1 in ECs. Our results show that BMK1 activation prevents EC apoptosis by stimulating phosphorylation of Bad in the cytoplasm and inhibiting caspase-3 activity. These data suggest a role for BMK1 in vascular homeostasis by inhibiting EC apoptosis.
Materials and Methods
Bovine lung microvascular ECs (BLMECs), endothelial basal medium, and supplements were purchased from Invitrogen Co. TNF-α was bought from Roche Diagnostics; CHX, MTT, and anti-BMK1 antibody were from Sigma Co. Hoechst 33342 was purchased from Molecular Probes Company. Goat anti-Actin, rabbit anti-Bcl-xL, anti-RSK1 polyclonal, and mouse anti-GST monoclonal antibodies were purchased from Santa Cruz. Mouse anti-Bad polyclonal antibody was from Transduction laboratories. Bcl-2 antibody was bought from Pharmingen Co. Anti-pBad (Ser112), anti-pBad (Ser136), anti-pAkt (Ser473), anti-pAkt (Thr308), anti-Akt, and anti-phospho-p90RSK (Thr359/Ser363) polyclonal antibodies were from Cell Signaling, Inc. Bad-wt and Bad-S112/136A constructs were generously given by Dr M. Greenberg (Harvard University, Boston, Mass).
Cell Culture, Transfection, Infection, and Flow Experiments
BLMECs are bovine lung microvascular endothelial cells. These cells have endothelial morphology similar to bovine aortic endothelial cells and share similar signaling transduction pathways, such as VEGF, TNF, and PDGF. BLMECs at passage 3 to 8 were grown in MCDB-131 media supplemented with 10% FBS, heparin (15300 U/L, Sigma), hydrocortisone (2.76 μmol/L, Sigma), bovine pituitary extract, epidermal growth factor (1.64 nmol/L, Sigma), l-glutamine, and antibiotics (100 U/mL penicillin, 68.6 mol/L streptomycin) in flasks precoated with 2% gelatin. For transient expression experiments, cells were transfected with plasmids or adenovirus 1 day after plating. After 24 hours of incubation, cells were treated with TNF-α and CHX, or serum deprived. MTT assay or nuclei staining was performed, or cells were harvested for Western blot analysis or caspase-3 activity assay. Flow experiments with cone and plate viscometer system were performed with cells grown in 60-mm dishes following our laboratory’s method.8 The experiments with the parallel-plate chamber system were performed with cells grown in 35-mm dishes and maintained in MCDB-131 media in a humidified atmosphere containing 5% CO2 at 37°C.
Determination of Cell Viability
Measurement of cellular MTT reduction was performed as described previously.22
Nuclear Morphology Analysis
Cells were stained with Hoechst 33342 and counted blindly. The percentage of apoptotic cells per total number of cells (GFP/RFP positive) was determined.
Detection of DNA Fragmentation by TdT-Mediated dUTP Nick-End Labeling (TUNEL)
Measurement of TUNEL fluorescence was performed following the manufacturer’s guide (Boehringer Mannheim Co).
Caspase-3-activity was detected by the color absorbance of the chromophore p-nitroanilide (pNA) obtained after proteolytic cleavage from the labeled substrate Asp-Glu-Val-Asp-pNA (DEVD-pNA) and pNA as standard using a spectrophotometer 405 nm (Clontech ApoAlert Caspase-3 assay kit).
Data are shown as mean±SE for 3 to 4 separate experiments. Differences were analyzed with one-way analysis of variance (ANOVA) or unpaired Student’s t test. Values of P<0.05 were considered statistically significant.
Fluid Shear Stress Activates BMK1 in BLMECs
We have shown that BMK1 is phosphorylated and activated by steady laminar fluid shear stress in bovine aortic ECs (BAECs).23 Because the transfection efficiency of BAECs or human umbilical vein ECs (HUVECs) is not high (usually ≤10%), we used BLMECs that have a much higher transfection efficiency (≈60%). BMK1 activation was measured by phosphorylation of BMK1 and MEF2C (substrate of BMK1). BMK1 was activated in BLMECs by steady fluid shear stress (12 dyne/cm2) in a time-dependent manner with peak at 40 minutes as shown by band shift due to BMK1 phosphorylation (Figure 1A) and peak at 20 to 40 minutes as shown by BMK1-MEF2C in vitro kinase assay (data not shown). The results are similar to those previously described in BAECs.23 Transfection with constitutively active MEK5 (CA-MEK5) strongly activated BMK1 (Figure 1B), but not other MAP kinases (data not shown).
BMK1 Activation Inhibits BLMECs Apoptosis Induced by Serum Deprivation
Previous studies indicated that BMK1 activation is associated with cell growth and survival.20,21 Because physiological fluid shear stress has been shown to protect ECs from apoptosis,7 BMK1 activation by fluid shear stress may contribute to the antiapoptotic effect of shear stress. To evaluate the role of BMK1 activation in endothelial cell viability, cell death was induced by serum deprivation. As shown in Figure 2A, cell viability decreased to 48±4% after incubation with 0% serum for 24 hours assayed by MTT. CA-MEK5 improved cell viability to 87±6% (n=3, P=0.01, Figure 2A). To prove that the survival effect of CA-MEK5 was due to decreased apoptosis, BLMEC apoptosis was measured by nuclear morphology (Figures 2B and 2C). Cells were cotransfected with eGFP in the presence or absence of CA-MEK5, then nuclei were stained with Hoechst 33342. Serum deprivation for 24 hours triggered 49% cell apoptosis indicated by nuclear condensation, whereas overexpression of CA-MEK5 significantly inhibited the apoptotic effect of serum deprivation (Figures 2B and 2C). Similar results were obtained with TUNEL analysis, as shown in Figure 2D, bottom.
Inhibition of BMK1 Activity With Dominant-Negative BMK1 Causes EC Death
To investigate the function of BMK1 in normal EC growth, BMK1 activity was inhibited by overexpressing DN-BMK1 (TEY to AEF mutation), which inhibits wild-type BMK1 activity.24 The viability of BLMECs transfected with DN-BMK1 decreased to 68% by MTT assay (data not shown) and apoptosis increased from 9.75% to 22% by nuclear morphology analysis, compared with control (Figure 3A). The results suggest that activity of BMK1 is important for maintaining endothelial cell viability through an antiapoptotic effect.
Inhibition of BMK1 Activity Prevented Shear Stress–Mediated Antiapoptotic Effect
Chronic steady laminar shear stress (24 hours) decreases EC apoptosis,7 and short-term shear stress (10 minutes) blocks TNF-mediated JNK activation.8 Because activation of BMK1 protects BLMECs from apoptosis, we next evaluated whether BMK1 is an important mediator for shear stress–mediated inhibition of apoptosis induced by serum deprivation by specifically inhibiting BMK1 with DN-BMK1. As shown by nuclear morphology analysis (Figure 3B) steady fluid shear stress for 1 hour significantly decreased BLMEC apoptosis in pcDNA transfected cells, compared with static condition (from 14.6% in static to 4.1% in flow). After overexpression of DN-BMK1, there was a 2-fold increase of apoptotic cells (from 14.6% to 28.1%) in static conditions, compared with pcDNA control, due to inhibition of basal BMK1 activity. The ability of shear stress to inhibit serum deprivation–induced apoptosis was markedly diminished by DN-BMK1 overexpression and was no longer significant (Figure 3B). Specifically, the protective effect of shear stress on apoptosis decreased from 71.9% protection in pcDNA transfected cells to 15.2% in DN-BMK1–transfected cells.
BMK1 Activation Protects Endothelial Cells From Apoptosis Induced by Inflammatory Stimuli
Numerous studies show inflammatory stimuli, such as TNF-α, are linked to induction of apoptosis. To determine whether BMK1 activity contributes to protection of endothelial cells from apoptosis induced by inflammatory stimuli, we investigated whether BMK1 activation inhibited endothelial cell apoptosis induced by TNF-α. As shown in Figure 3C, TNF-α and cycloheximide (CHX) (0.36 μmol/L) treatment induced endothelial cell death in a dose-dependent manner. Overexpression of CA-MEK5 increased cell viability dramatically. In contrast, inhibition of BMK1 activity by DN-BMK1 transfection augmented TNF- and CHX-induced cell death. Nuclear morphology showed apoptosis consistent with the change in cell viability (data not shown). In summary, BMK1 activity contributes to protection of endothelial cells from apoptosis induced by TNF-α.
We next determined whether BMK1 mediates protective effects of long-term fluid shear stress. BMK1 activity increased 2-fold after long-term flow (5 dyne/cm2) stimulation as measured by BMK1 reporter gene assay (data not shown). Nuclear morphology analysis (see online Figure 1 in the online data supplement available at http://www.circresaha.org) showed that steady shear stress (5 dyne/cm2) for 18 hours significantly decreased BLMEC apoptosis induced by TNF-α/CHX, compared with static condition (from 18.2% in static to 3.5% in flow). After overexpression of DN-BMK1, the ability of shear stress to inhibit TNF-α/CHX induced apoptosis was markedly diminished (from 30.8% in static to 19.8% in flow). On a percentage basis, the protective effect of shear stress decreased from 81.8% to 36.0% with overexpression of DN-BMK1, compared with pcDNA control (Figure 3D). There was a 1.7-fold increase in apoptosis after overexpression of DN-BMK1 (from 18.2% to 30.8%) in static condition, compared with control, due to inhibition of basal BMK1 activity. These data suggest that BMK1 is an important mediator for fluid shear stress to protect ECs from apoptosis.
Both serum deprivation and TNF-α/CHX induced EC apoptosis. Short-term (1 hour) or long-term (18 hours) steady shear stress activated BMK1 and inhibited EC apoptosis induced by serum deprivation or TNF-α/CHX similarly. To characterize the mechanism(s) by which BMK1 mediates the antiapoptotic effect of fluid shear stress, we used short-term flow to activate BMK1 and serum deprivation to induce apoptosis.
BMK1 Prevented EC Apoptosis by Inhibiting Caspase-3 Activity
Activation of the protease family of caspases represents the final common pathway of apoptosis signal transduction. Because shear stress has been suggested to interfere with activation of caspase-3 and thereby prevent apoptosis,7 we investigated whether activation of BMK1 reduced caspase-3 activity. Serum deprivation of BLMECs for 24 hours activated caspase-3 ×5.2-fold. Activation of BMK1 by overexpressing CA-MEK5 significantly decreased caspase-3 activity (Figure 4A).
Bad Phosphorylation by Fluid Shear Stress Is Required to Protect ECs From Apoptosis
The mechanism for the antiapoptotic effect of BMK1 was studied by measuring expression and phosphorylation of several common apoptosis-regulating proteins. Expression of Bad, Bcl-2, or Bcl-xL was not changed by activation of BMK1 with CA-MEK5 (Figure 4B). However, phosphorylation of Bad on Ser136, required for the association of Bad with 14-3-3 (which inhibits apoptotic signaling), was increased by overexpression of CA-MEK5 (Figure 4B, top). Similarly, Bad phosphorylation on Ser112 was also increased (data not shown).
We further investigated whether fluid shear stress altered phosphorylation of Bad. As shown in Figure 4C, exposure of BLMECs to steady flow for 30 minutes increased Bad phosphorylation on Ser136 by ≈4-fold. Phosphorylation was to an extent similar to that observed with CA-MEK5 (Figure 4C, compare lane 2 with lane 3). Phosphorylation of Bad by flow was significantly blocked by overexpression of DN-BMK1. Bad Ser112 was also phosphorylated by fluid shear stress (data not shown). It is well known that phosphorylation of Bad on Ser136 and Ser112 is important for association with 14-3-3, which then sequesters Bad in the cytoplasm, thereby preventing translocation to mitochondria and apoptosis. To evaluate the role of Bad phosphorylation in the antiapoptotic effect of BMK1 activation, we cotransfected Bad-wt (wild type) or Bad-S112/136A with CA-MEK5 or pcDNA as control plasmid. Bad-wt overexpression induced apoptosis in 15.9% of cells and CA-MEK5 overexpression decreased apoptosis to 7.9%. The protective effect of CA-MEK5 overexpression on apoptosis was abolished in cells that overexpressed Bad-S112/136A (from 25.6% to 31.1%) (Figure 4D and online Figure 2 in the online data supplement). Similar results were obtained when apoptosis was assayed by annexin V-FITC/PI FACS (online Figure 3 in the online data supplement). Specifically, CA-MEK5 inhibited apoptosis induced by Bad-wt by 64% (from 22.2% apoptosis in pcDNA-transfected cells to 8.0% apoptosis in CA-MEK5–transfected cells), but had no significant effect on apoptosis induced by Bad-S112/S136A (from 31.5% apoptosis in pcDNA-transfected cells to 36.0% apoptosis in CA-MEK5–transfected cells). These data strongly suggest that the antiapoptotic effect of fluid shear stress is mediated by activation of BMK1, phosphorylation of Bad, and sequestration of Bad in the cytoplasm.
Bad Phosphorylation Mediated by Fluid Shear Stress and BMK1 Is Independent of Akt, PKA, or p90RSK Activity
Because sequence analysis of mouse Bad protein showed no mitogen-activated protein kinase (MAPK) consensus recognition sequence located at Ser112 or Ser136, Bad phosphorylation by BMK1 may be indirect. There are several candidate kinases including Akt, PKA, and p90RSK that have been reported to phosphorylate Bad on Ser136 or Ser112. Akt was already known to be a Bad-Ser136 kinase and has been shown to mediate antiapoptotic effects of fluid shear stress.12 Therefore, we investigated the role of Akt in BMK1-regulated Ser136 phosphorylation of Bad. As shown in Figure 5A, Akt was activated by fluid shear stress, measured by phosphorylation of Ser473 and Thr308 of Akt. CA-MEK5 overexpression did not stimulate phosphorylation of the two sites, and DN-BMK1 did not inhibit phosphorylation of Akt induced by flow. LY294002, a specific inhibitor of Akt activity, decreased Akt phosphorylation induced by flow (Figure 5B), but did not inhibit Bad phosphorylation at Ser136 induced by BMK1 activation via overexpression of CA-MEK5 (Figure 5B, bottom). These observations indicate that Akt is not involved in the regulation of Bad phosphorylation by BMK1 activity.
Another candidate mediator is PKA because PKA was reported to regulate Bad phosphorylation on Ser112 and flow stimulated PKA.25 To measure PKA activation, we measured VASP phosphorylation, a well-known PKA substrate.26 VASP is a 46-kDa protein phosphorylated preferentially at Ser157 by PKA, which leads to a shift from 46 to 50 kDa on SDS-PAGE and is detectable with anti-VASP antibody. Forskolin activated PKA as shown by VASP mobility shift (Figure 5C, top, lane 3). Forskolin-induced PKA activation was blocked by adenoviral overexpression of the specific inhibitor, PKI, as shown by decreased VASP phosphorylation (Figure 5C, top, lane 4). However, PKI expression did not inhibit Bad Ser112 phosphorylation induced by flow (Figure 5C, bottom, compare lanes 2 and 4). These data indicate that PKA does not mediate Bad phosphorylation by flow.
Finally, the role of p90RSK as a mediator was studied. p90RSK activity was measured by phosphorylation at Thr359 and Ser363 (which are critical for the activity).27 Flow increased p90RSK phosphorylation (Figure 5D), but phosphorylation was not inhibited by DN-BMK1 overexpression. Also, overexpression of CA-MEK5 did not phosphorylate these sites (Figure 5D). These data indicate that p90RSK is not the mediator for Bad phosphorylation by BMK1. Therefore, the data suggest BMK1 activation regulates Bad phosphorylation via an unknown mediator(s), independent of Akt, PKA, or p90RSK activity.
The major findings of this study are that BMK1 protects endothelial cells from apoptosis induced by growth factor deprivation and incubation with proinflammatory stimuli, and BMK1 is an important mediator for the antiapoptotic effect of steady fluid shear stress. The antiapoptotic mechanism of shear stress and BMK1 activation likely involves phosphorylation of Bad on Ser136 and Ser112 because increasing BMK1 activity (by either overexpression of CA-MEK5 or exposure to flow) stimulated Bad phosphorylation and inhibited apoptosis. In contrast, overexpression of DN-BMK1 inhibited phosphorylation of Bad and stimulated apoptosis. Although BMK1 has been reported to be important for cell survival in several cell types, the present study is the first to identify BMK1 as a mediator for the antiapoptotic effect of fluid shear stress, and to show that Bad is a target for BMK1.
The mechanisms by which fluid shear stress protects ECs from apoptosis are largely unknown. Because MAPK pathways coordinate activation of gene transcription, protein synthesis, cell cycle machinery, differentiation, and cell death,28 MAPKs are logical candidates to mediate the antiapoptotic effects of flow. Several findings support a specific role for BMK1 relative to other MAPKs. The BMK1 knockout mice display defects in vessel formation and cardiac development with abnormal EC morphology.14,15 In contrast, no cardiovascular defects were reported with ERK1/2 knockout mice.29 In addition, the activation of ERK1/2 by flow in ECs is transient compared with BMK1.23 Finally, the other two ubiquitous MAPKs (p38 and JNK) are not highly activated by shear stress, especially compared with their activation by proinflammatory stimuli such as TNF.8
Our findings suggest that BMK1 activation by flow is very important for protection of ECs from apoptosis because DN-BMK1 overexpression increased EC apoptosis to 19.5%, even in the presence of serum (data not shown). Long term flow was shown by Dimmeler and colleagues7 to reduce HUVEC apoptosis induced by growth factor withdrawal or TNF-α treatment. In this study, we show that flow for 1 hour at 24 dyne/cm2 inhibited apoptosis induced by serum deprivation, and flow for 18 hours at 5 dyne/cm2 protected BLMECs from apoptosis induced by TNF-α/CHX. The antiapoptotic effect of flow was significantly inhibited by overexpression of DN-BMK1. Two recent studies of the BMK1 knockout mouse demonstrated that BMK1 is important for angiogenesis and embryonic cardiovascular development. Because the defects in the BMK1 knockout occurred at the onset of blood flow (day 9.5), we may speculate that increased apoptosis occurred due to loss of BMK1.
We propose two mechanisms for the antiapoptotic effect of BMK1 activity: regulation of apoptotic gene expression and posttranslational modification of proteins involved in apoptosis. Suzaki et al21 suggested that BMK1 (via MEF2C) regulated gene expression and increased PC12 cell survival. However, they did not identify the apoptosis-related genes involved. In the present study, we found no change in expression of Bcl-2, Bcl-xL, and Bad in response to BMK1 activation by overexpression of CA-MEK5. However, Bad phosphorylation was significantly increased. Bad is reported to be phosphorylated on Ser136, Ser112, and Ser155.30–33 Bad phosphorylation by BMK1 may be direct, but no MAPK consensus recognition sequences were found at Ser136 and Ser112. Thus it is possible that kinases downstream of BMK1 such as Akt, PKA, and p90RSK may be directly responsible for Bad phosphorylation. Akt has been reported to be involved in fluid shear stress–dependent EC survival12 and can phosphorylate Bad on Ser136.34 Other kinases such as p90RSK and PKA can directly phosphorylate Bad on Ser112 only, or both Ser112 and Ser155, respectively. However, inhibiting Akt, PKA, and p90RSK had no effect on BMK1-mediated Bad phosphorylation. Thus, the downstream mediators of BMK1 that regulate Bad phosphorylation require further investigation.
We propose a model (Figure 6) to explain how BMK1 mediates antiapoptotic signals induced by flow. Activation of MEK5 and BMK1 leads to Bad phosphorylation on Ser136 and Ser112. Phosphorylation promotes association of Bad with the scaffold protein 14-3-3. Binding of 14-3-3 with Bad sequesters Bad in the cytoplasm and keeps it from translocating to mitochondria where it binds to Bcl-xL and induces cytochrome C release and activation of caspase-3. Important questions to be answered are how flow activates MEK5 and how BMK1 promotes phosphorylation of Bad on Ser136 and Ser112. It is likely that the antiapoptotic effects of steady laminar shear stress involve several mechanisms. Previously flow was shown to increase Akt phosphorylation, and to upregulate expression of antiapoptotic genes Bcl-xL and the soluble antagonistic Fas isoform FasExo6Del.35 Shear stress also activates the VEGF receptor 2 (VEGF-R), which likely inhibits apoptosis.36,37 We found that inhibiting VEGF-R activity with a blocking antibody or with a specific drug inhibitor did not block flow-mediated BMK1 activation (data not shown), suggesting that VEGF-R is not involved in BMK1 signaling in ECs. In conclusion, BMK1 is a novel mediator of the antiapoptotic effect of fluid shear stress (independent of VEGF-R, Akt, PKA, and p90RSK) that stimulates phosphorylation of Bad.
This work was supported by HL49192 and HL64839 to B.C.B. We thank Drs Abe, Aizawa, and Jin for very useful discussions. We are grateful to Dr Fujiwara, Dr Abe, and Tamlyn Thomas for technical support and use of the parallel-plate chamber apparatus.
Original received November 22, 2002; first resubmission received June 9, 2003; second resubmission received November 17, 2003; accepted December 3, 2003.
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