Specific Induction of tie1 Promoter by Disturbed Flow in Atherosclerosis-Prone Vascular Niches and Flow-Obstructing Pathologies
Nonlaminar flow is a major predisposing factor to atherosclerosis. Yet little is known regarding hemodynamic gene regulation in disease-prone areas of the vascular tree in vivo. We have determined spatial patterns of expression of endothelial cell receptors in the arterial tree and of reporter gene constructs in transgenic animals. In this study we show that the endothelial cell–specific receptor Tie1 is induced by disturbed flow in atherogenic vascular niches. Specifically, tie1 expression in the adult is upregulated in vascular bifurcations and branching points along the arterial tree. It is often confined to a single ring of endothelial cells functioning as sphincters and hence experiencing the steepest gradient in shear stress. In aortic valves, tie1 is asymmetrically induced only in endothelial cells encountering changes in flow direction. Disturbance of laminar flow by a surgical interposition of a vein into an artery led to induction of tie1, specifically in the region where the differently sized vessels adjoin. In pathological settings, tie1 expression is specifically induced in areas of disturbed flow because of the emergence of aneurysms and, importantly, in endothelial cells precisely overlying atherosclerotic plaques. Hemodynamic features of atherosclerotic lesion-prone regions, recreated in vitro with the aid of a flow chamber with a built-in step, corroborated an upregulated tie1 promoter activity only in cells residing where flow separation and recirculation take place. These defined promoter elements might be harnessed for targeting gene expression to atherosclerotic lesions.
The luminal surface of blood vessels is constantly exposed to hemodynamic forces, primarily to shear stress that is the tangential force engendered on endothelial cell surfaces by the blood flow. Hemodynamic forces play a fundamental remodeling role in the vascular network during its formative stage as well as in adapting the adult vasculature to pathophysiological changes in shear stress (eg, the process of arteriogenesis1). Hemodynamic forces are also a considerable factor in the development of vascular pathologies such as atherosclerosis, aneurysms, poststenotic dilatations, and arteriovenous malformations. Notably, these diseases have the propensity to develop in vascular regions distinguished by disturbed flow that occurs naturally in certain vascular niches. Atherosclerosis, although clearly associated with some systemic risk factors, is a geometrically focal disease that preferentially develops at the outer edges of blood vessel bifurcations and at points of blood flow recirculation and stasis (eg, in aortic valves). In these predisposed locations, fluid shear stress on the vessel wall is significantly lower in magnitude and exhibits directional changes and flow separation, features absent from regions of the vascular tree generally spared from atherosclerosis.2
The detrimental effects of disturbed flow have inspired extensive research to elucidate the transduction processes by which endothelial cells convert these mechanical stimuli into biochemical signals and to identify the molecular mediators. These studies, including high-throughput DNA microarray analyses, uncovered a large number of shear stress–regulated genes that, together, function in multiple regulatory pathways in endothelial cells.3,4 These studies, however, were mostly carried out in cultured endothelial cells, ie, in the absence of the normal regulation exerted by matrix components and possibly also by circulating factors and without the cross-talk with periendothelial cells. Furthermore, because it is difficult to recreate in vitro the complex flow patterns prevailing in atherogenic microenvironments of the vessel wall, it is not known which pairwise comparison of shear conditions is of relevance in vivo. Although more recent in vitro studies have better approximated the hemodynamic features of atherosclerosis-prone regions,5,6 in situ analysis of gene expression in the native context of atherosclerosis-prone areas has been limited. Upregulated gene expression in atherosclerosis-prone areas has been demonstrated for the junction molecule connexin43,7 vascular cell adhesion molecule (VCAM)-1,8 and certain transcription factors.9,10
We have used a candidate gene approach in conjunction with in situ hybridization analysis of the native arterial tree and of vessels with flow-obstructing pathologies. To uncover promoters induced in predisposed vascular niches, we have also examined reporter gene constructs in transgenic mice and rats. Endothelial cell–specific receptors are natural candidates for sensing changes in hemodynamic forces and for participating in the mechanotransduction process. Recent studies showing that shear stress induces a rapid phosphorylation of endothelial transmembrane receptors and their concomitant association with adapter proteins in the absence of a ligand11–13 have additionally focused our attention on endothelial tyrosine kinase receptors.
The subject of this study is the endothelial cell–specific, tyrosine kinase receptor Tie1. Despite intensive efforts, a ligand for Tie1 has not been found. Yet Tie1 is indispensable for maintaining vascular integrity, and its absence results in vessel rupture and hemorrhages.14,15 Although Tie1 is abundantly expressed in the embryonic vasculature, expression grossly subsides concomitantly with vessel maturation. However, previous studies16 and findings reported below have shown that constitutive expression of tie1 is maintained in selected areas of the adult vasculature, suggesting that Tie1, in addition to its established role in stabilizing embryonic vessels, might also play a role in maintaining proper vascular function in the adult.
In this study we show that tie1 expression is selectively induced in areas constantly exposed to disturbed flow, ie, the areas prone to develop atherosclerosis. Moreover, we show that tie1 is focally upregulated as a consequence of pathological flow obstructions, including by the protrusion of atherosclerotic lesions into the lumen. These findings, in conjunction with the identification of promoter elements sufficient to target expression of a surrogate gene to areas of disturbed flow, suggest the potential utility of tie1 promoter in targeting expression of therapeutic gene products to atherosclerotic lesions.
Materials and Methods
Tie1-lacZ transgenic mice were previously described.17 Tie-lacZ transgenic rats (outbred Wistar strain HsdBrl:WH) were generated by the standard pronuclear microinjection technique using the same construct that was used in the production of Tie-lacZ transgenic mice. The zygotes were transferred into the oviducts of the pseudopregnant recipient females immediately after microinjection. The transgenic line UKUR11 was selected for additional studies and bred to homozygosity. Apolipoprotein (Apo) E–deficient mice18 on a C57BL/6 background were fed Western diet, and 6- to 9-month-old animals were monitored for the appearance of aortic lesions.
Epigastric vein (EV) to common femoral artery (CFA) interposition grafts were performed in rats as previously described.19 Briefly, male Sabra rats (220 to 420 g) were anesthetized, and the CFA and EV were exposed. The superficial circumflex iliac branch of the CFA was isolated, and a 1-cm-long segment of the artery was resected. A 1-cm-long segment of the ipsilateral EV was harvested, irrigated with heparinized saline solution, and installed as a reversed interposition graft. Total ischemic time was <90 minutes, and graft patency was confirmed by visual inspection. Vein grafts were harvested along with the flanking aortic segments at day 67 after operation and fixed in 4% paraformaldehyde. At the time of harvest, grafts remained patent and with no detectable thrombi.
In Situ Hybridization
Paraffin-embedded aortas and heart caps from ApoE-deficient mice were sectioned, processed, and hybridized in situ as previously described using 35S-labeled riboprobes.20 Tie1-specific and vascular endothelial growth factor receptor (VEGF-R2)–specific probes were amplified from the respective cDNAs and cloned into the TOPO-PCRII vector (Invitrogen) using the following primers: Tie1, 5′-CCTGGGCCCTGCCTCACCC-3′ and 5′-GGGGGGGCGCTCATAGGGC-3′; VEGF-R2, 5′-GTGCAGGATGGAGAGCAAGG-3′ and 5′-TGGACTCAATGGGCCTTCCATTTCTGTACC-3′.
Tissues from transgenic rats and mice were fixed in buffered 4% paraformaldehyde. Tissues were then washed three times for 30 minutes at room temperature in PBS (pH 7.4) containing 2 mmol/L MgCl2, 0.02% NP40, and 0.01% Na-deoxycholate and stained overnight at 37°C in the same buffer supplemented with 5 mmol/L potassium-ferricyanide, 5 mmol/L potassium-ferrocyanide, and 1 mg/mL X-gal. Specimens were then fixed in formalin, embedded in paraffin, sectioned, and counterstained with H&E or, alternatively, examined as whole mounts.
Immunostaining of Whole-Mount Retina Preparations
X-gal–stained retinas were washed in PBS and permeabilized in 0.5% Triton/PBS for 2 hours before adding α-smooth muscle actin antibody (Sigma) and overnight incubation at 37°C with gentle shaking. After five 1-hour washes in PBS, a secondary anti-mouse antibody conjugated to HRP (Amersham) was added in 0.5% Triton/PBS/1% BSA and retinae-incubated for 3 hours at room temperature. Specimens were then stained with 3-amino-9-etylcabazole (Sigma), mounted on glass slides, and viewed in a light microscope.
Flow Apparatus Generating Laminar Flow or Disturbed Flow
A parallel plate chamber, designed in the laboratory of Yedgar and colleagues, as previously described,21 was used. Briefly, a silicon rubber gasket was sandwiched between two plastic plates, creating a gap of 250 μm, and a slide covered with test cells was inserted in a hole drilled in the lower plate. The chamber was connected to a recirculating flow circuit composed of a peristaltic pump, a pressure transducer, and a reservoir with culture medium maintained at 37°C, pH 7.4, and gassed with 5% CO2 and 95% air. Flow velocity was adjusted to 20 mL/min, generating shear stress of calculated 12 dyne/cm2. To generate conditions of flow separation, recirculation, and reattachment, a similar apparatus was designed, except that the slide on top of which cells were seeded contained in its middle a 200-μm-high, descending step (see Figure 6A). Confluent cultures of primary bovine aortic endothelial cells at a low passage number (<10) on gelatin-coated slides were subjected to flow and subsequently analyzed as described below.
Infection of Endothelial Cells With a pTie-Luciferase Adenovirus and In Situ Mapping of Luciferase Activity
The same 735-bp-long segment of the tie1 promotor used in the transgenic experiments (see below) was used to generate a Tie1 promoter-luciferase adenovirus (also containing a cytomegalovirus promoter–driven green fluorescent protein by the pAdTrack system22). For infection, cells were preincubated in the absence of serum for 1 hour and then infected at a multiplicity of infection of 50 to 100 plaque-forming units per cell.
To determine total luciferase activity, cells were scraped off the slide and lysed, and luciferase activity quantified using a dual detection kit (Promega) according to the manufacturer’s instructions. Redistribution of luciferase activity after flow was determined by incubating the same cell-seeded slide with luciferin for 5 minutes at room temperature (both before and after flow) and in situ detection of emitted light in each case with the aid of a cooled CCD camera (Roper chemiluminescence imaging system, model LN/CCD-1300EB), as described by Honigman et al.23 The integrated light acquired during a short exposure was presented as pseudo-color images (blue, least intense; red, most intense), and relative quantification of luciferase activity in successive fields was performed using the software supplied with the cooled charge-coupled device (CCCD) camera.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Upregulation of tie1 Promoter Activity in Aortic Flow Dividing Points
Reasoning that hemodynamic regulation of tie1 will be reflected in a nonuniform expression along the arterial vascular tree, we mapped sites of upregulated tie1 expression along the arterial system. To provide a convenient way for spatial resolution, we used tie1 promoter β-galactosidase reporter transgenic mice and rats. The transgenic animals used contained 735 bp of proximal mouse promoter sequences cloned upstream of the reporter gene. Previous studies have shown that this segment of the promoter is sufficient to confer a pattern of expression similar to that of the endogenous gene.16,17 The arterial tree was isolated from adult animals and analyzed as a whole-mount preparation for β-galactosidase activity. Strikingly, β-gal activity was mostly restricted to arterial segments located at vascular junctions and downstream of bifurcations and branching points, ie, at sites experiencing shear stress gradients or constantly exposed to a disturbed flow (Figure 1). Notably, tie1 is strongly expressed at the aorta-renal artery junction, a site distinguished by its almost straight branching angle and, consequently, a site particularly exposed to disturbed flow and exceedingly prone to atherosclerosis (Figure 1, inset).
Upregulated tie1 Expression in Microvascular Branching Points and Capillary Sphincters
Anticipating that induction of tie1 expression at sites exposed to disturbed flow will be manifested also in the microvasculature, we analyzed tie1 promoter activity in the vascular network of the retina. To make sure that the observed pattern of expression is dictated by the promoter sequences rather than being effected by the position of transgene integration, both transgenic rats (Figure 2, top) and transgenic mice (Figure 2, bottom) harboring the same tie1 promoter- β-galactosidase transgene were examined. In both species, tie1 promoter activity was found to be strongly upregulated at points of primary and secondary branching from main arterioles. It is predominantly upregulated in capillary endothelial cells residing just downstream of the branch point, ie, in cells experiencing the steepest gradient in shear stress. Interestingly, these endothelial cells are also functionally distinguished from their neighbors with regard to their increased association with vascular smooth muscle cells (VSMCs), qualifying them as capillary sphincters (see inset in bottom image). Specific induction of tie1 in capillary sphincters was not limited to the retina and was also visible in the microvasculature of other tissues, like skin (data not shown).
Asymmetric Expression of tie1 in Aortic Valve Endothelium
The aortic and pulmonary artery valves (also known as the semilunar valves) represent a unique niche within the cardiovascular system with respect to the hemodynamic forces exerted on the endothelium. Hydraulic models, simulating the aortic valve, have demonstrated flow recirculation and eddy currents that swirl behind the flexible cusps during rapid flow through the valve orifice.24 In the aortic outlet, β-gal activity was restricted to the valves and was undetectable in the vessel wall downstream (Figure 3A). Sectioning through the outflow tract has shown that tie1 promoter is exclusively expressed in endothelial cells lining the inner aspect of the cup-shaped cusps but not in endothelial cells at the outer aspect of the same leaflet (Figures 3B and 3C). This unique pattern of expression was specific for tie1 and was not detected in VEGF-R2-lacZ mice (data not shown). This asymmetrical pattern of expression strongly argues for hemodynamic regulation, ie, that flow conditions experienced only by these cells are conducive for tie1 induction.
Specific Induction of tie1 Expression in Atherosclerotic Lesions
To determine whether flow obstruction induced by luminal protrusion of atherosclerotic lesions will result in tie1 induction, we carried out an in situ hybridization analysis determining the spatial distribution of endogenous tie1 mRNA in plaque-bearing ApoE-null mice. As shown in Figure 4, tie1 mRNA was dramatically upregulated in the endothelium overlying atherosclerotic plaques. Remarkably, the area of upregulated tie1 expression (quantified as 8-fold increase) precisely colocalized with the lesion area and was sharply demarcated from the nonexpressing flanking healthy endothelium. Upregulated tie1 expression was observed already in early, small, and relatively acellular lesions (Figures 4A and 4B) and persisted in advanced plaques (Figures 4C and 4D).
This unique pattern of expression was specific to tie1 and was not observed with other endothelial cell–specific tyrosine kinase receptors, such as VEGF-R2 (Figure 4F), VEGF-R1, and tie2 (data not shown). A closer inspection of the endothelial surface revealed that the tie1-expressing cells, particularly those positioned almost perpendicular to the direction of flow, have rearranged into a tile-like configuration (Figure 4E).
Atherosclerotic lesions developed in the aortic sinuses of ApoE-null mouse fed Western diet were also examined for in situ tie1 expression. As shown in Figures 4G and 4H, tie1 was also induced in the endothelium overlying lesions at this particularly atherogenic site.
Induction of tie1 Expression by Disturbed Flow in Aneurysms and Vein Grafts
Atherosclerotic lesions represent a complex situation where hemodynamic regulation might be compounded by the effect of different cytokines produced in the underlying plaque. We wished, therefore, to examine a situation of disturbed flow in an otherwise healthy endothelium. Abdominal aneurysms spontaneously developed in aged tie1-lacZ transgenic animals were examined for tie1 promoter expression. Although β-gal–positive cells were never detected in the same region of the healthy aorta, the tie1 promoter was strongly upregulated downstream of the aneurysmal dilatation (see online Figure 1S, available in the online data supplement at http://www.circresaha.org).
To examine whether tie1 expression is upregulated when laminar flow is disturbed by means of a surgical manipulation, vein-to-artery interposition grafts were performed as described in Materials and Methods. The grafts were harvested more than 2 months after surgery to assure that any induced trauma had been resolved and possible endothelial cell injuries had been repaired. Reasoning that a complex pattern of disturbed flow would be created near the junctions where vessels of a markedly different caliber are adjoined, we subjected the grafts to an in situ hybridization analysis with tie1. As shown in Figure 5, expression of the endogenous tie1 gene was indeed induced, preferentially in vein endothelial cells close to the A/V junction. Notably, expression of tie1 mRNA was not significantly upregulated in the more distal vein endothelium, suggesting that the pattern of disturbed flow presiding close to the junction is responsible for upregulated expression.
Tie1 Is Negatively Regulated by Shear Stress In Vitro
The particular sites of upregulated tie1 expression have in common areas of reduced shear stress or even stasis. Therefore, expression of tie1 was determined in vitro under conditions of stasis and at different times after the onset of laminar flow conditions that engender the endothelial cell monolayer with a shear stress of ≈12 dyne/cm2. Negative regulation of tie1 expression by shear stress was indeed demonstrated for both tie1 mRNA and TIE1 protein as well as for tie1 promoter activity (see online Figure 2S).
Spatial Mapping of tie1 Promoter Activity in a Step-Flow Chamber Simulating Disturbed Flow
To better approximate the in vivo situation, we aimed to recreate in vitro a gradient of fluid shear stress and other flow phenomenon present in atherosclerosis-prone areas, particularly flow separation and recirculation. To this end, a flow chamber apparatus was used in which endothelial cells are seeded on top of a slide with a central backward-facing (ie, descending) step (see Materials and Methods and Figure 6A). Previous studies using a similar device have demonstrated its utility in modeling major hemodynamic factors present in atherosclerotic lesions.5,6,25 To spatially correlate modulations in tie1 promoter activity with graded variations in hemodynamic factors experienced by endothelial cells at different locations, a tie1 promoter-luciferase reporter was used in conjunction with in situ mapping of luciferase activity. Briefly, endothelial cells were preinfected with an adenovirus vector encoding a tie1 promoter-driven luciferase, and a uniform infection of >90% cells was demonstrated with the aid of a cytomegalovirus promoter–driven green fluorescent protein reporter contained in the same vector. A constant flow was then applied for 16 hours to allow sufficient time for reaching a hemodynamic steady state and for gene expression reprogramming. The spatial distribution of luciferase activity was determined with the aid of a sensitive light-detection CCCD camera and software for producing pseudo-color images depicting relative light intensities in situ (see Materials and Methods). Before the onset of flow, a uniform distribution of luciferase activity was observed (Figure 6C). Exposure of the same culture to a disturbed flow, however, led to redistribution of luciferase activity, depending on the position of cells relative to the point where laminar flow has been disturbed (Figure 6D). Most pronounced upregulated activity was confined to a narrow band of cells located just downstream of the step. Remarkably, this region fully coincided with the area distinguished by flow separation and recirculation (Figure 6B), as deduced from previous studies where streamlines were calculated for a similar backward-facing step apparatus. For example, in an apparatus with identical geometry (ie, step height of 200 μm), the point of flow reattachment at the right hand end of the vortices was mapped at ≈300 μm downstream of the step.25 As evident from the quantification shown in Figure 6E, luciferase activity in the region of disturbed flow was ≈4-fold higher than in its flanking regions. Notably, the difference in laminar shear stress existing upstream and downstream of the step had only a slight effect on promoter activity, thus highlighting the role of hemodynamic factors associated with flow disturbance rather than a mere change in wall shear stress as the key factor in promoter induction.
This study represents a comprehensive in vivo analysis of a hemodynamically regulated promoter with respect to its specific induction in restricted niches within the arterial tree and in important vascular lesions associated with an obstructed flow. Findings reported in this study show that tie1 is dramatically and specifically upregulated in areas of disturbed flow coinciding with atherogenic vascular niches. Moreover, it is shown that tie1 expression is induced on experimental and pathological obstruction of blood flow, including in emerging atherosclerotic plaques and in areas of disturbed flow generated by the development of aneurysms.
This in vivo approach has several clear advantages over commonly used approaches to the study of hemodynamically regulated promoters using cultured endothelial cells. First, it integrates regulatory cues exerted by all components of the vessel wall in their natural contexts, as well as systemic influences. Second, expression profiles are determined under the authentic, complex hemodynamic features presiding in each and every vascular niche, a situation that is difficult to recreate in vitro. It should be pointed out, however, that although this approach precisely maps the native sites of upregulated expression, it may come short in determining the responsible flow parameter (eg, distinguishing disturbed shear stress from a shear stress gradient). Third, because of its superior spatial resolution, the in situ methodology enables pinpointing at endothelial cells experiencing conditions conducive for promoter activation at the resolution level of single cells. The latter is exemplified in showing that upregulated tie1 expression is often confined to a single ring of endothelial cells, namely, in capillary sphincters. It should be pointed out, however, that it could not be determined whether tie1 was induced by the particular flow pattern at sphincters or, alternatively, by the cyclic strain exhibited by VSMCs on sphincter constriction or by the action of VSMC-secreted factor. Also, it remains to be determined whether signaling through the Tie1 receptor plays a role in augmented VSMC recruitment.
The specific locales of tie1 expression do not reflect organ specificity, do not distinguish arteries from veins, and are independent of vessel caliber. Instead, the common denominator of locales showing upregulated tie1 expression is some disturbance of a uniform shear stress. Two vascular configurations are particularly noticeable for a high degree of disturbed flow as well as for being most atherogenic: branching points with a large branching angle (eg, aortic branching to the kidney) and sites constantly experiencing flow reversal (eg, aortic valves). These locales were indeed identified as the sites of most-pronounced tie1 induction (Figures 1 and 3⇑, respectively). The most compelling evidence for the claim that disturbed flow is the trigger for tie1 upregulation was obtained from surgical manipulation of vein interpositioning, where tie1 mRNA was specifically induced at sites distinguished by an abrupt change in vessel diameter (Figure 5).
Certain vascular pathologies introduce disturbances in an otherwise laminar flow or aggravate an already existing disturbance, notably, atherosclerotic plaques and regional dilatations associated with aneurysms. As reported here, these lesions indeed led to focal induction of tie1 expression. Hemodynamic induction of tie1 may also explain previous results of its upregulation in arteriovenous malformations.26 Thus, elevated tie1 expression not only marks atherosclerosis-prone areas but is also an indicator of vascular lesions associated with obstructed flow. A similar situation has been previously reported for VCAM-1. Specifically, elevated levels of VCAM-1 expression were observed in endothelial cells in regions of the mouse ascending aorta and arch that are predisposed to lesion formation, and, furthermore, VCAM-1 was also expressed by endothelial cells in early atherosclerotic lesions and by intimal cells in more advanced lesions.8 The latter is different from the case of tie1, where expression remained exclusive to the endothelium overlying the lesion, also in advanced lesions.
The function of Tie1 and the significance of Tie1 signaling in endothelial cell biology are poorly understood, primarily because of the failure to find a ligand. Yet a signal transduction pathway for tie1 was recently elucidated.27 It was also demonstrated that proteolytic shedding of the putative ligand-binding exodomain of Tie1 generates a truncated receptor still capable of ligand-independent signaling.28,29 Interestingly, a recent study has shown that downregulation of tie1 in endothelial cells exposed to shear stress in vitro is also accompanied by receptor cleavage.30 Thus, precedents for a ligand-independent mechanotransduction in other endothelial-specific tyrosine kinase receptors11–13 might also apply for tie1 expressed in vascular microenvironments experiencing disturbed flow.
The notion that signaling through Tie1 might be required for the endothelium to sustain hemodynamic forces is consistent with vascular rupture in tie1-null mice and with findings that Tie1 signaling induces an antiapoptotic response.27 Consistent with a role for tie1 signaling in the recruitment of periendothelial cells is our observation of a correlation between upregulated tie1 expression in capillary sphincters and their association with a higher number of vascular smooth muscle cells (Figure 2).
The hemodynamically regulated expression patterns described above were generated using a defined segment of promoter sequence. Specifically, 735 bp of the tie1 promoter was sufficient to confer a tight hemodynamic regulation on a surrogate gene both in vivo and in vitro. This is the first transgenic study demonstrating hemodynamic regulation dictated by a defined regulatory element. A recent in vitro study has delineated a negative shear stress response element to a 250-bp element within the tie1 promoter.30
Irrespective of the unknown function of Tie1, findings reported here might have important implications solely based on the unique features of its promoter. Conceivably, the critical promoter sequences might be used for targeting expression of any gene of interest to atherosclerotic plaques. For example, targeting expression of a gene that will promote plaque passivation might have a significant benefit. Experiments examining the feasibility of harnessing the tie1 promoter for targeting expression to atherosclerotic plaques through the use of intra-arterially delivered viral vectors are ongoing.
This work was supported by a grant from the Israel Science Foundation. We thank Dr H. Giladi for adenovirus vectors, E. Zeira for in situ luciferase analysis, Dr S. Yedgar for advice on flow devices, and R. Sinervirta and A. Järvinen for technical help.
↵*Both authors contributed equally to this study.
Original received July 1, 2003; first resubmission received September 16, 2003; second resubmission received October 31, 2003; revised second resubmission received November 24, 2003; accepted December 1, 2003.
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