Role of Caspases in Ox-LDL–Induced Apoptotic Cascade in Human Coronary Artery Endothelial Cells
Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL–induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 μg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1β–converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL–induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL–induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.
Low-density lipoprotein (LDL) is oxidized in the subendothelial space of the arterial wall to become a highly injurious product, oxidized low-density lipoprotein (ox-LDL), which results in vascular endothelial injury.1 Several lines of evidence have suggested that ox-LDL plays a critical role in the pathogenesis of atherosclerosis and destabilization of the atherosclerotic plaque,2 myocardial ischemia/reperfusion injury,3 and acute myocardial infarction.4 Vascular endothelial cells in vitro and in vivo internalize and degrade ox-LDL through a receptor-mediated pathway, which mainly involves the lectin-like endothelial ox-LDL receptor (LOX-1).5 Recent studies from our laboratory and other groups have shown that LOX-1 expression in endothelial cells is upregulated by several stimuli including ox-LDL,5 cytokine tumor necrosis factor-α (TNF-α),6 and angiotensin II7 as well as fluid shear stress.8
Apoptosis, also called programmed cell death, is an important process of many pathological conditions including atherosclerosis. Ox-LDL can induce apoptosis in a variety of tissues and cells including human coronary artery endothelial cells (HCAECs),5 smooth muscle cells,9 and macrophages.10 The initiation and regulation of apoptosis is highly controlled by a family of proteolytic enzymes, the caspases. The activity of different caspases is controlled by specific apoptotic proteins, which either stimulate or inhibit their activity. The upregulation of proapoptotic proteins, such as Fas, and the downregulation of antiapoptotic proteins, such as Bcl-2, have been shown to play a critical role in the activation of apoptotic caspase signaling pathway.11
It is known that Bcl-2 can prevent the release of mitochondrial cytochrome c into cytosol, which is required to activate caspase-9 and caspase-3, and then induce apoptosis.12 Therefore, increased Bcl-2 expression is widely believed to inhibit apoptosis. C-IAP (inhibitory apoptotic protein)-1 is an inhibitor of procaspase-9 and procaspase-313 and is inactivated by the release of Smac from mitochondria. On the other hand, Fas, a membrane receptor of Fas ligand, can induce caspase-8 and then caspase-3 activation, which is suppressed by c-FLIP.14 The present study was designed to study the role of ox-LDL and its receptor LOX-1 in the modulation of proapoptotic and antiapoptotic proteins, activation of caspase pathways and induction of apoptosis in HCAECs.
Materials and Methods
The initial batch of HCAECs was purchased from Clonetics Corp. The endothelial cells were pure, based on morphology and staining for factor VIII and acetylated LDL. These cells were 100% negative for smooth muscle cell α-actin expression. HCAECs were cultured as described earlier.1,5 HCAECs were incubated with different concentrations of ox-LDL (10 to 80 μg/mL) for different periods of time (2 to 24 hours) to study the expression of Bcl-2, c-IAP-1, Fas, and c-FLIP, the truncation of Bid, and the release of cytochrome c and Smac from mitochondria. The concentration and incubation time of ox-LDL for maximal effects were used to conduct the following studies.
To determine the role of LOX-1 in the expression or release of these apoptotic proteins, HCAECs were pretreated with antisense to LOX-1 mRNA (0.5 μmol/L) or sense (0.5 μmol/L) for 48 hours, and then HCAECs were exposed to ox-LDL (80 μg/mL) for 24 hours. The cells were then harvested to determine the expression or release of proteins mentioned above. HCAECs pretreated with antisense- or sense-LOX-1 were also used to examine ox-LDL–mediated activation of caspase-3, -8 and -9 and apoptosis. In parallel experiments, HCAECs were pretreated with the caspase-3 inhibitor DEVD-CHO (25 μmol/L), the caspase-8 inhibitor z-IETD-fmk (25 μmol/L), or the caspase-9 inhibitor z-LEHD-fmk (25 μmol/L) followed by treatment with ox-LDL (80 μg/mL) for 24 hours, and then the cells were harvested to determine the activity of caspases and apoptosis. The concentration of these reagents was based on previous studies.15,16
Preparation of Antisense and Sense to LOX-1 mRNA
Antisense phosphorothioate oligonucleotides and sense phosphorothioate oligonucleotides (as control) directed to the 5′-coding sequence of the human LOX-1 mRNA were designed and manufactured by Biognostik GmbH. Hereafter, the antisense and sense to LOX-1 mRNA will be referred to as antisense-LOX-1 and sense-LOX-1, respectively. The antisense-LOX-1 was synthesized as a 16-mer product (8 bases) targeted at 5′-CAGTTAAATGAGCCCG-3′ of the LOX-1 mRNA sequence. The corresponding control (sense) was 16-mer (8 bases) targeted at 5′-ACCTACGTGACTACGT-3′. Logarithmically growing endothelial cells were transfected by adding antisense-LOX-1 or sense-LOX-1 into culture medium according to the instructions of the manufacturer. The detailed method for preparation and use of antisense- and sense-LOX-1 has been described in our publications.5,17
Preparation of Lipoproteins
Native LDL and ox-LDL were prepared as described earlier.1 In brief, human native LDL was isolated from human blood plasma by discontinuous centrifugation. LDL was oxidized by exposure to CuSO4 (5 μmol/L free Cu2+ concentration) in PBS at 37°C for 24 hours. The thiobarbituric acid–reactive substance content of ox-LDL was 10.2±0.28 versus 0.56±0.16 nmol per 100 μg protein in the native LDL preparation (P<0.01). LDL and ox-LDL were kept in 50 mmol/L Tris-HCl, 0.15 mol/L NaCl and 2 mmol/L EDTA at pH 7.4 and were used within 10 days of preparation.
Western Blot Analysis of Apoptotic Proteins in HCAECs
HCAEC lysates from each group (45 μg protein per lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After incubation in blocking solution (5% nonfat milk, Sigma Chemical Co), membranes were incubated with 1 μg/mL primary antibodies overnight at 4°C. Antibodies used were mouse monoclonal antibody to Bcl-2, rabbit polyclonal antibody to c-FLIP, rabbit polyclonal antibody to Fas (Oncogene), rabbit polyclonal antibody to c-IAP-1 (Santa Cruz Biotech), rabbit polyclonal antibody to Bid (BD Biosciences), mouse monoclonal antibody to cytochrome c (BD Biosciences), rabbit polyclonal antibody to β-actin (Sigma Chemical), and rabbit polyclonal antibody to Smac (a gift from Dr Xiaodong Wang, Drexel University, Philadelphia, Pa). Membranes were washed with 1× TBST solution and then incubated with secondary antibody (1:5000 dilution, Amersham Life Sciences) for 2 hours. The membranes were detected with the ECL system (Amersham Life Sciences) and relative intensities of protein bands were analyzed by Scan-gel-it software.7
Measurement of Apoptosis by TUNEL Staining
To detect DNA fragmentation in situ, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) assay was performed as previously described.5,18 Briefly, the cells plated on slides were fixed with 4% methanol-free formaldehyde, pH 7.4, for 25 minutes at 4°C and washed with PBS. The slides were incubated with 0.2% Triton X-100 for 5 minutes on ice to increase cell permeability and were equilibrated with terminal deoxynucleotidyl transferase (TDT) buffer (including 30 mmol/L Tris-HCl, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) for 10 minutes at room temperature. The slides were covered with 0.3 U/μL TDT and 0.04 nmol/μL fluorescein-12-dUTP (Promega) in TDT buffer for 60 minutes at 37°C. The slides were immersed in 2×SSC buffer for 15 minutes at room temperature and then washed with PBS to remove unincorporated fluorescein-12-dUTP. The slides were immersed in 1.5 μg/mL of propidium iodide in PBS for 15 minutes at room temperature and washed with deionized water. The slides were viewed under a fluorescence microscope with green fluorescence set at 520 nm and red fluorescence set at >620 nm. The cells stained with green color indicate apoptotic cells. At least 500 cells from randomly selected fields in each sample were counted to determine the percentage of apoptotic cells.
DNA Fragmentation Gel Electrophoresis (DNA Laddering)
The results of TUNEL staining were further confirmed by DNA laddering. In brief, the HCAECs were removed from culture dishes, washed twice with PBS, and pelleted by centrifugation. Cell pellets were then treated for 10 minutes with lysis buffer (1% NP-40 in 20 mmol/L EDTA and 50 mmol/L Tris-HCl, pH 7.5) on ice. After centrifugation for 5 minutes at 1600g, the supernatant was collected, and the extraction was repeated with the same amount of lysis buffer. The supernatants were brought to 1% SDS and treated for 2 hours with RNase A (final concentration 5 μg/μL) at 56°C, followed by digestion with proteinase K (final concentration 2.5 μg/μL) for 2 hours at 37°C. After addition of 1/2 vol of 10 mol/L ammonium acetate, the DNA was precipitated with 2.5 vol of absolute ethanol. DNA was recovered by centrifugation at 12 000g for 10 minutes and dissolved in gel loading buffer. DNA was separated by electrophoresis in 1.5% agarose gel containing ethidium bromide.5,18
Measurement of Caspase-3, Caspase-8, and Caspase-9 Activity
The activities of caspase-3, caspase-8, and caspase-9 were measured according to the kit manufacturers’ instructions. In brief, HCAECs were removed from culture dishes, washed twice with PBS, and pelleted by centrifugation. Cell pellets were then treated for 10 minutes with iced lysis buffer supplied by the manufacturers: Caspase-3 cellular activity assay kit (Calbiochem), Caspase-8 assay kit (Oncogene), and Caspase-9 assay kit (Calbiochem). Then the suspensions were centrifuged at 10 000g for 10 minutes, and the supernatants were transferred to a clear tube. To each tube, specific substrate conjugate [acetyl-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD- p-NA) for caspase-3, acetyl-Ile-Glu-Thr-Asp-aminotrifluoromethyl coumarin (Ac-IETD-AFC) for caspase-8 and acetyl-Leu-Glu-His-Asp-p-nitroaniline (Ac-LEHD-p-NA) for caspase-9] was added and the tubes were incubated at 37°C for 2 hours. During incubation, the caspases cleaved the substrates to form p-NA or AFC. Caspase-3 and -9 activities were read in a microtiter plate reader at 405 nm. Caspase-8 activity was read in a fluorescent plate reader at 400 nm for excitation and at 505 nm for emission.
All data represent the mean of duplicate samples from at least 3 separately performed experiments. Data are presented as mean±SD. Statistical significance was determined in multiple comparisons among independent groups of data in which ANOVA and the F test indicated the presence of significant differences. A value of P<0.05 was considered significant.
Ox-LDL, LOX-1, and Expression/Release of Apoptotic Proteins in HCAECs
Ox-LDL treatment decreased the expression of Bcl-2 and c-IAP-1 in a concentration- and time-dependent fashion (Figure 1). Also, ox-LDL treatment, especially at 80 μg/mL concentration for 24 hours, induced an increase in the release of mitochondrial activators of apoptosis, cytochrome c, and Smac (Figure 2). These effects of ox-LDL on Bcl-2 and c-IAP-1 expression as well as cytochrome c and Smac release were almost completely blocked by antisense-LOX-1. In contrast, sense-LOX-1 had no effect (Figure 3). Interestingly, ox-LDL treatment did not change the expression of Fas and c-FLIP. Likewise, pretreatment of cells with antisense- or sense-LOX-1 had no effect on the expression of Fas and c-FLIP. Further, ox-LDL treatment also did not change the level of Bid truncation in the cytoplasm (data not shown).
Ox-LDL, LOX-1, and Activation of Caspase Pathways in HCAECs
We observed ox-LDL–mediated activation of both caspase-9 and caspase-3, but not caspase-8 (Figure 4). Further, compared with ox-LDL alone, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk (25 μmol/L) inhibited ox-LDL–induced activation of caspase-9 and caspase-3 (both P<0.01). Pretreatment of HCAECs with the caspase-3 inhibitor DEVD-CHO (25 μmol/L) only inhibited ox-LDL–induced activation of caspase-3, but not caspase-9. In parallel experiments, pretreatment of HCAECs with the caspase-8 inhibitor z-IETD-fmk (25 μmol/L) had no effect on caspase-3 activation elicited by ox-LDL (Figure 4). Notably, caspase-8 activity fell significantly with caspase-8 inhibitor treatment showing the appropriate effect of the inhibitor. We also identified that pretreatment of HCAECs with the inhibitor of either caspase-8 or caspase-9 did not affect ox-LDL–induced release of cytochrome c and Smac from mitochondria (Figure 5).
It is noteworthy that the effects of ox-LDL on caspase pathway were mediated by its receptor LOX-1, because pretreatment of HCAECs with the antisense-LOX-1 reduced ox-LDL–induced activation of caspase-9 and caspase-3, whereas sense-LOX-1 had no such effect (Figure 6).
Ox-LDL and Apoptosis in HCAECs
Ox-LDL treatment increased the percentage of apoptotic cells (from 7.6±2.5% to 27.3±2.7%; P<0.01). Preincubation of HCAECs with antisense-LOX-1 decreased the percentage of apoptotic cells from 27.3±2.7% to 7.9±2.2% (Figure 7). In contrast, sense-LOX-1 had no such effect on ox-LDL–induced apoptosis. This effect of ox-LDL on apoptosis of HCAECs and the role of LOX-1 in this effect were further confirmed by DNA laddering (Figure 7).
In parallel studies, pretreatment of HCAECs with the caspase-9 inhibitor decreased ox-LDL–induced apoptosis (from 31.4±4.3% to 8.8±1.8%; P<0.01), whereas caspase-8 inhibitor had no effect (Figure 8). These results were confirmed by DNA laddering (Figure 8). TUNEL staining and DNA laddering also showed that caspase-3 inhibitor almost completely blocked ox-LDL–induced apoptosis in HCAECs (Figure 8).
In the present study, we found that ox-LDL decreased the expression of antiapoptotic proteins Bcl-2 and c-IAP-1 in a concentration- and time-dependent manner. In addition, ox-LDL enhanced the activity of caspase-9 and then caspase-3. The activation of these caspases was secondary to the release of mitochondrial activators of caspases, ie, cytochrome c and Smac. Ox-LDL did not affect the expression of proapoptotic protein Fas and antiapoptotic protein c-FLIP. There was also no activation of caspase-8 and Bid truncation, indicating absence of a role of caspase-8 pathway. These data also point against a cross-talk between mitochondria-dependent caspase-9 pathway and mitochondria-independent caspase-8 pathway in ox-LDL–induced apoptosis in endothelial cells. These effects of ox-LDL were mediated by its endothelial receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1 almost completely blocked the effects of ox-LDL. Overall, these data indicate that ox-LDL via activation of LOX-1 reduces antiapoptotic proteins and causes the release of activators of caspases with resultant activation of caspase pathway leading to apoptosis in HCAECs.
Ox-LDL, LOX-1, Apoptotic Proteins, and Caspase Pathway
Increasing evidence suggests that ox-LDL plays a critical role in endothelial injury, myocardial ischemia,3,19 and atherosclerosis.1–4 LOX-1, a newly described ox-LDL receptor, is expressed largely in endothelial cells.5–8,20 Studies from our and other laboratories have shown that LOX-1 mediates ox-LDL uptake by endothelial cells, monocytes adhesion to activated endothelial cells,17 and apoptosis of endothelial cells5 as well as cardiac myocytes.3 Other studies have shown that apoptosis of endothelial cells is involved in the progressing of atherosclerosis and rupture of atherosclerotic plaques.1–5,9–11 A study from our laboratory showed that ox-LDL through LOX-1 induces apoptosis of HCAECs that relates to the activation of mitogen-activated protein kinases and nuclear factor-κB.5 In another study, we found that ox-LDL activates protein kinase C (β subunit) and then increases the expression of metalloproteinases and collagenase activity.21 These observations suggest that ox-LDL and its receptor LOX-1 are crucial in the evolution of atherosclerosis and its complications. However, the mechanism of ox-LDL–induced apoptosis and atherosclerosis is far from clear. In the present study, we attempted to further investigate the steps leading to apoptosis in HCAECs in response to ox-LDL.
It is known that Bcl-2 is an antiapoptotic protein that can inhibit apoptosis induced by a mitochondria-dependent caspase-9 pathway.22 Bcl-2 protein has been shown to prevent apoptosis induced by diverse stimuli by inhibiting the mitochondrial release of cytochrome c that stimulates caspase-9 activity or by acting as an antioxidant23 or by mechanisms unrelated to its effect on reactive oxygen radicals.10 Overexpression of the Bcl-2 gene with the use of gene transfer techniques inhibits apoptosis in rat smooth muscle cells24 and murine aortic endothelial cells.25 Presently, we show that ox-LDL decreases Bcl-2 expression and that the effect of ox-LDL on Bcl-2 expression is mediated by LOX-1, because the pretreatment with antisense-LOX-1 blocked the effect of ox-LDL on Bcl-2 expression. In this study, the involvement of Bcl-2 was further confirmed by the finding that ox-LDL increased the release of cytochrome c from mitochondria and that ox-LDL significantly induced caspase-9 and caspase-3 activity. Because caspase-9 inhibitor had no effect on ox-LDL–induced cytochrome c release, it suggested that cytochrome c functions upstream of caspase-9.
C-IAP-1 is another antiapoptotic factor that can inhibit apoptosis. It belongs to IAP family that can inhibit cytochrome c–induced proteolytic processing and activation of procaspase-9 and procaspase-3.13 By preventing the activation of these caspases, c-IAP-1 exerts antiapoptotic effect. Therefore, the increased expression of c-IAP-1 suppresses apoptosis, and conversely, the decreased expression of c-IAP-1 stimulates apoptosis.13 The present study showed that ox-LDL decreases c-IAP-1 protein expression in a concentration- and time-dependent fashion. We also found that the effect of ox-LDL on c-IAP-1 protein expression is mediated by LOX-1, because the pretreatment with antisense-LOX-1 blocked the effect of ox-LDL on c-IAP-1 expression. In addition, after ox-LDL treatment, there was a marked release of Smac from mitochondria into the cytoplasmic compartment that prevented c-IAP-1 from exerting protection against caspase-9 and caspase-3 activation. Thus it appears that ox-LDL affects not only the expression of c-IAP-1, but also the function of c-IAP-1. Both of these effects favor apoptosis. Further, our present study provides clear evidence that Smac functions upstream of caspase-9, because caspase-9 inhibitor had no effect on ox-LDL–induced Smac release.
Fas is a cell surface receptor for Fas ligand and a cell surface initiator of cell death.26 Activating Fas by the binding of Fas ligand activates a cascade of cysteine aspartate-specific proteases, the caspases (especially, caspase-8 and caspase-3) that result in apoptosis.27 During this process, the death domain of Fas interacts with an intracellular adapter protein Fas-associated death domain (FADD), which then recruits and activates the initiator procaspase-8 (previously called FLICE).28 After activation, caspase-8 can activate procaspase-3 and induce apoptosis. We wondered whether ox-LDL can induce Fas expression and cause apoptosis in HCAECs. Interestingly, we found that ox-LDL did not affect Fas expression and caspase-8 activity. Lack of Bid truncation further supports the concept of lack of caspase-8 activation, because Bid is a substrate that is truncated by active caspase-8 to form truncated Bid (tBid).
C-FLIP is a FLICE-inhibitory protein. Fas ligand-Fas interaction leads to the recruitment of the adaptor protein FADD and the inactive form of cysteine protease procaspase-8 (FLICE), and gives rise to the death-inducing signaling complex (DISC), in which procaspase-8 is activated. The activation of caspase-8 is regulated by the cellular FLICE-inhibitory protein (c-FLIP). C-FLIP can be recruited to DISC and block further recruitment of procaspase-8 into the complex, thereby inhibiting the proteolytic activation of caspase-8 and suppressing apoptosis.28 In this study, we observed that ox-LDL did not affect the antiapoptotic protein c-FLIP expression. Consistently, we noted lack of activation of caspase-8 in response to ox-LDL, as discussed in the previous paragraph. These findings collectively suggest against the involvement of mitochondria-independent caspase-8–mediated pathway in ox-LDL–induced endothelial injury.
Caspase-3, also named as CPP32, is a widely expressed protease and is considered an executioner protease in cells during apoptosis. It is known that caspase-3 is a cysteine protease that cleaves poly(ADP-ribose) polymerase (PARP), nuclear lamins, gelsolin, and others. Under normal conditions, caspase-3 is present as an inactive proenzyme in live cells. Several upstream molecules of caspase-3, such as caspase-8 and caspase-9, can activate procaspase-3 by proteolytic cleavage. In this study, we confirmed that caspsae-9 functions upstream of caspase-3, because pretreatment of cells with the caspase-9 inhibitor reduced caspase-3 activation, whereas caspase-3 inhibitor pretreatment had no effect on caspase-9 activation. The activated caspase-3 abrogates the effects of several substrates that protect cellular integrity such as PARP, gelsolin, actin, lamins, fodrin, focal adhesion kinase, and DNA fragment factor.29 As a result, apoptosis occurs.
We also demonstrated that ox-LDL induces activation of caspase-3 through its receptor LOX-1, which relates to the decrease in Bcl-2 and c-IAP-1 expression, in the release of cytochrome c and Smac and activation of caspase-9. LOX-1 plays a critical role in this process since antisense-LOX-1 inhibited the effects of ox-LDL on modulation of antiapoptotic proteins and activation of caspase-9 as well as caspase-3. These findings provide critical information on ox-LDL–induced signaling pathway of apoptosis in endothelial cells.
LOX-1 and Ox-LDL–Induced Apoptosis
Previous studies from our laboratory have demonstrated that ox-LDL treatment induces apoptosis in HCAECs, and this effect is mediated via LOX-1.5,18 In the present study, we confirmed this phenomenon. Importantly, we found that the ox-LDL–induced apoptotic signaling pathway includes the downregulation of antiapoptotic proteins c-IAP-1and Bcl-2, stimulated release of cytochrome c and Smac, activation of caspase-9 and caspase-3, and finally induction of apoptosis. On the other hand, ox-LDL does not affect Fas, c-FLIP, and caspase-8 pathway in HCAECs. These findings provide new insights into the role of LOX-1 in ox-LDL–mediated endothelial injury.
This work was supported by grants from a Scientist Development Grant from the American Heart Association and a Merit Review Award from the Department of Veterans Affairs.
Original received August 26, 2003; resubmission received November 17, 2003; revised resubmission received December 3, 2003; accepted December 10, 2003.
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