Cooling-induced contraction and protein tyrosine kinase activity of isolated arterioles in secondary Raynaud's phenomenon

P. B. Furspan, S. Chatterjee1, M. D. Mayes2 and R. R. Freedman

Department of Psychiatry and Behavioral Neurosciences, Wayne State University, Detroit, MI 48201, 1 Department of Rheumatic and Immunologic Diseases, The Cleveland Clinic Foundation, Cleveland, OH 44195 and 2 Department of Internal Medicine (Rheumatology), University of Texas Houston Health Science Center, Houston, TX 77030, USA.

Correspondence to: P. Furspan, C.S. Mott Center, Wayne State University, 275 E. Hancock Ave., Detroit, MI 48201, USA. E-mail: aa1985{at}wayne.edu


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Objective. To investigate the response of skin arterioles from control subjects and patients with scleroderma and Raynaud's phenomenon (RP/SSc) to cooling and modulators of protein tyrosine kinase (PTK) activity.

Methods. We used the microvessel perfusion technique to characterize the response of isolated dermal arterioles (100–200 µm, outside diameter) from normal (n = 17) and RP/SSc (n = 17) subjects to cooling from 37° to 31°C. Fluorescent immunohistochemistry was used to measure tyrosine phosphorylation.

Results. Arterioles from control subjects exhibited dilation in response to cooling from 37 to 31°C whereas those from RP/SSc subjects contracted (+4.3 ± 1.7 vs –16.7 ± 3.1%, P<0.05, n = 6). In the presence of the protein tyrosine phosphatase inhibitor sodium orthovanadate (SOV, 10 µM), the response of arterioles from control subjects did not change; however, arterioles from RP/SSC subjects exhibited a significantly greater contraction (–72.6 ± 19.7%; P<0.05, n = 6). Tyrosine phosphorylation of arterioles at 37°C from control and RP/SSc subjects was similar. In response to cooling to 31°C, however, arterioles from RP/SSc subjects exhibited a significantly greater increase in tyrosine phosphorylation compared with those from control subjects (43 ± 7.0% vs 10 ± 3.8%; P<0.01). SOV increased tyrosine phosphorylation in arterioles from both groups (73 ± 11.6% vs 42 ± 5.6%; P<0.05, n = 5). Arterioles from RP/SSC subjects precontracted with norepinephrine exhibited greatly attenuated relaxation to acetylcholine compared with those from control subjects.

Conclusion. The results of this study support the view that the hallmark of Raynaud's phenomenon associated with scleroderma, cooling-induced vasospasm, appears to be mediated by an increase in PTK activity possibly exacerbated by impaired endothelium-dependent vasodilation.

KEY WORDS: Cooling, Vasospasm, Raynaud's phenomenon, Vascular smooth muscle, Signal transduction, Endothelium, Scleroderma


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Exposure to cooling is the primary trigger of vasospasm in individuals with Raynaud's phenomenon (RP) associated with scleroderma (RP/SSc). The aetiology and pathophysiological mechanism underlying this hyper-responsiveness to cooling and the reason for its frequent association with scleroderma remain obscure. Recent studies investigating the biochemical basis of cold-induced modification of vascular response suggest a possible mechanistic link between RP/SSc and protein tyrosine kinase (PTK) activity [1–3]. Wagerle et al. demonstrated that the cold-induced contraction of the middle cerebral artery of the lamb could be reversed by inhibitors of PTK, but not by a protein kinase C inhibitor [1]. Conversely, sodium orthovanadate (SOV), a broad-spectrum inhibitor of protein tyrosine phosphatases (enzymatic inactivators of PTK) potentiated the cold-induced contraction. Recently, we reported that skin arterioles isolated from patients with primary RP (PRP) contracted in response to cooling from 37° to 31°C whereas those isolated from control subjects did not [4]. The cooling-induced contraction was reversed in a concentration-dependent manner by the PTK inhibitors genistein and tyrphostin 47. Also, using fluorescent immunohistochemistry and an antiphosphotyrosine antibody, we determined that there is greater tyrosine phosphorylation in response to cooling in arterioles from PRP subjects than in those from control subjects.

Here we sought to determine if increased PTK activity also plays a role in the vasospasm associated with secondary Raynaud's phenomenon. We therefore investigated the effect of mediators of PTK activity on cooling-induced contraction and tyrosine phosphorylation of isolated skin arterioles from RP/SSc patients and control subjects.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Subject characteristics
Seventeen patients with RP/SSc (14 women, three men) and 17 normal volunteers (15 women, two men) served as subjects. Average age was 44 ± 5.3 yr for the RP/SSc subjects. Scleroderma (systemic sclerosis) was defined according to the American College of Rheumatology criteria [5]. Average duration of the disease was 13.2 ± 3.4 yr. Nine patients had the limited form of the disease and eight had the diffuse form. All patients had Raynaud's phenomenon, defined as episodic, bilateral, digital colour changes (two out of three possible colours: blanching, cyanosis, rubor) provoked by cold and/or emotional stress. The patients were recruited from a registry of scleroderma patients in Michigan administered by one of us (M.D.M.) and sponsored by the National Institutes of Health. No patient had active digital ulcers. Subjects were not hypertensive or hypercholesterolaemic. Medications taken by the patients varied: methotrexate in two; gastrointestinal medications, lansoprazole in two, cisapride in one, omeprazole in one; lipid-regulating agents, gemfibrozil in one; thyroid hormones, levothyroxine in three; diuretics, triamterene in one; calcium antagonists, nifedipine in three; blood flow enhancers, pentoxifylline in two; cholinergic agents, pilocarpine in one. All medications were withdrawn at least 1 week prior to the study. All were non-smokers.

The normal volunteers (average age 31.7 ± 10.6 yr, not significantly different from mean patient age) were recruited from signs posted on our medical campus requesting subjects for research on blood vessels. They were screened by being giving a medical history and completing an extensive symptom questionnaire. They were free of all medication and none smoked. All patients and volunteers gave written informed consent according to the World Medical Association Declaration of Helsinki. All procedures and the design of the study also were approved by the Institutional Review Board of the Wayne State University Human Investigation Committee.

Vessel preparation
Using lidocaine (without norepinephrine) as a local anaesthetic, skin biopsies (6 mm in diameter) were taken from the medial forearm of normal and RP/SSc subjects. In the latter, biopsies were taken from clinically uninvolved skin, as determined by the rheumatologist performing the procedure. Biopsies were pinned skin side down in a silicone-coated dissecting dish and covered with physiological salt solution (in mM):, NaCl, 118; KCl, 4.7; KH2PO4, 1.18; MgSO4-7H2O, 1.17; CaCl2-2H2O, 1.6; NaHCO3, 25.0; dextrose, 5.5; and CaNa2 EDTA, 1.2. Using a dissecting microscope (30x magnification), extra-fine forceps and microsurgical scissors were used to remove fat and connective tissue to uncover arterioles lying in the dermal–subcutaneous boundary. Arterioles of usable size (mean pressurized diameter 133 ± 9 and 130 ± 7 µm in control and RP/SSc subjects, respectively), distinguished from venules by their greater stiffness and refractive properties, were then carefully dissected out from the surrounding tissue. Vessels were used the same day or stored in Eagle's minimum essential medium (Sigma, St Louis, MO, USA) overnight at 4°C. Preliminary experiments found no difference in reactivity between vessels used the same day and vessels from the same biopsy used the next day. Similar results have been reported in the literature [6, 7]. One end of an arteriole was attached to the proximal micropipette using individual strands from 3–0 nylon braided suture and superfused with physiological salt solution aerated with 95% O2 and 5% CO2 (Fig. 1). A perfusion pressure of ~20 mmHg was used to clear the lumen of blood followed by attachment of the distal end of the artery segment to the closed distal pipette. The superfusate was warmed to and maintained at 37°C. Transmural pressure was controlled with a pressure-servo system. After an equilibration period of 45 min at 20 mmHg the pressure was increased to 40 mmHg [8]. Precise and rapid (<2 min) temperature reduction was accomplished by cooling the superfusate as it flowed through a jacketed temperature exchange coil connected to a second circulating heating/cooling water bath maintained at 31°C. All vessels were initially exposed to physiological salt solution containing 95 mM KCl (equimolar substitution for NaCl).



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FIG. 1. Schematic representation of the perfused microvessel apparatus. The pressure-servo, peristaltic pump and pressure transducer serve to maintain a constant pressure in the no-flow configuration of the arteriole. The arteriole is immersed in superfusate that is constantly flowing and to which agents of interest are added. Drug dilution is based on 50 ml, the volume of superfusate contained in the tubing, reservoir and chamber. The arteriole is visualized by a video camera attached to an inverted microscope. Arteriolar diameter is determined using a video dimension analyser, the output of which is fed into a computer.

 
Contractile activity of the blood vessels was monitored with a video camera and quantified with a video dimension analyser (Living Systems Instrumentation, Burlington, VT, USA) connected to a video monitor (Fig. 1). Changes in diameter were recorded on videotape and a computer-based data acquisition system (Dataq Instruments, Akron, OH, USA) and expressed as a percent change from baseline.

Experimental protocols
Perfused microvessel
The response of vessels to cooling from 37° to 31°C was determined in the absence and presence of SOV (10 µM), an inhibitor of tyrosine phosphatase, added 10 min before cooling. Genistein (1–100 µM) or tyrphostin 47 (0.1–10 µM) was added after contraction occurred and had reached a plateau for at least 5 min. Each concentration of the inhibitor was allowed to reach a steady state for at least 5 min before the addition of the next higher concentration.

After precontraction with norepinephrine (10–6 M) had reached a plateau for at least five min, increasing concentrations of acetylcholine (10–9 to 10–6 M) were added to the bath. At each concentration the effect of acetylcholine was allowed to reach a steady state for at least 5 min before the addition of the next higher concentration.

All results were expressed as percentage change from baseline lumen diameter.

Fluorescent immunohistochemistry [9]
Freshly obtained dermal arteries 1–3 mm in length and 100–200 µm in diameter were cleaned of adventitial tissue and cut into pieces ~200 µm in width. Segments were placed in wells containing 50 µl of phosphate-buffered saline (PBS) composed of (in mM) 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8 Na2HPO4. After incubation in PBS at 37°C on a thermostatically controlled block for 30 min, the pieces were transferred to wells containing PBS and the agents of interest. The temperature of selected wells was reduced to 31°C by adding an equivalent volume of 25°C PBS and, if called for, the agent of interest. After 5 min at the appropriate temperature the vessel segments were transferred to PBS containing 2% paraformaldehyde for 10 min to fix the tissues (this and subsequent steps were performed at room temperature). Next, the tissue was incubated with 0.1% Triton X-100 in PBS for 10 min. After a wash in PBS the pieces were incubated in a solution containing 0.9% Na-citrate, 2% goat serum, 1% bovine serum albumin, 0.05% Triton X-100, 0.025% NaNO3 and tetramethyl rhodamine isothiocyanate (TRITC)-labelled phosphotyrosine monoclonal antibody (1:50; Sigma) for 45 min. After incubation, the vessel pieces were washed with 0.02% Triton X-100 and 0.9% Na-citrate in PBS and then in PBS alone. The tissue segments were then mounted on glass slides and examined under a microscope equipped with an epifluorescence optical system. Measurement of fluorescence intensity as an indicator of tyrosine phosphorylation was performed using SimplePCI image analysis software (Compix, Imaging Systems, Cranberry Township, PA, USA).

Data analysis
Results were analysed by one-way analysis of variance supported by the Bonferroni test when pairwise between-group comparisons were performed. In all cases, a P value less than 0.05 denoted statistical significance between groups. All results were expressed as mean ± S.E.M.

Drugs
All drugs and chemicals were obtained from Sigma, St Louis, MO, USA. Genistein and tyrphostin 47 were dissolved in dimethyl sulphoxide (DMSO).


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
We observed no structural differences between arterioles from the two groups.

Arterioles from control and RP/SSC subjects contracted similarly to 95 mM KCL (–85 ± 2.5 vs –78 ± 5.4%, n = 12).

When the bath temperature was reduced from 37° to 31°C, untreated arterioles from control subjects dilated slightly whereas those from RP/SSc subjects contracted modestly (+4.3 ± 1.7 vs –16.7 ± 3.1%, P<0.05, n = 6) (Figs 2 and 3). Pretreating the arterioles with the protein tyrosine phosphatase inhibitor SOV had no effect on baseline diameter of arterioles in either group at 37°C. SOV did not change the response of arterioles from control subjects to cooling but did increase the contraction of arterioles from RP/SSc subjects (+3.2 ± 2.1 vs –72.6 ± 19.7%, P<0.05, n = 6) (Figs 2 and 3). Cumulative addition of genistein to the arterioles from RP/SSc patients treated with SOV caused a concentration-dependent decrease in contraction (Fig. 4). Tyrphostin 47 had a similar effect (data not shown). The SOV-modified contraction in response to cooling of one artery from an RP/SSc subject differed from the others in pattern and degree. In this artery the typical relatively gradual contraction was followed by a very rapid contraction that completely collapsed the lumen of the vessel (Fig. 5). This contraction was almost entirely reversed by cumulative addition of genistein (Fig. 5).



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FIG. 2. Representative tracings illustrating the effect of cooling from 37° to 31°C on arterioles from control and RP/SSc subjects in the absence and presence of 10 µM SOV, an inhibitor of protein tyrosine phosphatase.

 


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FIG. 3. Graphic summary of the data from experiments examining the effect of cooling from 37° to 31°C on the contractile response of arterioles from control and RP/SSc subjects in the absence and presence of 10 µM SOV, an inhibitor of protein tyrosine phosphatase. The response of the arterioles from RP/SSc subjects in the absence and presence of SOV was significantly greater (P<0.05) than those from control subjects. Sample size is 6.

 


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FIG. 4. Effect of genistein on the contraction of skin arterioles from RP/SSc subjects that was induced by reducing the bath temperature from 37° to 31°C in the presence of 10 µM SOV. Sample size is 6.

 


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FIG. 5. Tracing showing the pronounced response to cooling of an individual arteriole treated with 10 µM SOV from an RP/SSc subject. The pattern of contraction is reminiscent of vasospasm: gradual contraction followed by rapid and complete collapse of the lumen. The relaxant effect of the PTK inhibitor genistein suggests a role for this pathway in the contraction.

 
Fluorescent intensity did not differ between the two groups at 37°C (control, 25.5 ± 1.3 vs RP/SSc, 29.7 ± 3.9 arbitrary units; n = 6). Cooling increased tyrosine phosphorylation in arteriole segments from control and RP/SSc subjects (Figs 6 and 7). The increase in the segments from RP/SSc subjects, however, was significantly greater than in those from control subjects (43 ± 7.0 vs 10 ± 3.8%; P<0.05, n = 6). SOV increased tyrosine phosphorylation to a similar extent in both groups at 37°C (RP/SSc, 26.9 ± 1.8% vs control, 26.2 ± 7.7%; not significant, n = 5) (Fig. 7), whereas in response to cooling arterioles from RP/SSc subjects exhibited a greater increase in phosphorylation than those from control subjects (73 ± 11.6 vs 42 ± 5.6%; P<0.05, n = 5) (Figs 6 and 7). Genistein prevented the increase in tyrosine phosphorylation in response to cooling (Figs 6 and 7). Tyrphostin 47 had a similar effect (data not shown). DMSO (1:10 000 dilution), the vehicle for SOV, genistein and tyrphostin 47 was without effect on contraction or phosphorylation (data not shown).



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FIG. 6. Photomicrographs of arteriole segments treated invarious ways and then exposed to TRITC-conjugated antiphosphotyrosine antibody.

 


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FIG. 7. Change in tyrosine phosphorylation (as indicated by fluorescent intensity) of skin arteriole segments from control and RP/SSc subjects in response to SOV (10 µM) at 37°C and to decreasing the bath temperature from 37° to 31°C in the absence and presence of SOV (10 µM) and genistein (30 µM). Sample size is 5 for each bar. *P<0.05 compared with control subject value; #P<0.05 compared with untreated value.

 
Arterioles precontracted with 10–6 M norepinephrine from control (–91 ± 4.5%, n = 6) and RP/SSc (–84 ± 7.9%, n = 6) subjects exhibited significantly different responses to the endothelium-dependent vasodilator acetylcholine. As illustrated in Fig. 8, arterioles from RP/SSc subjects were relatively insensitive to acetylcholine compared with those from control subjects.



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FIG. 8. Concentration–response curves to acetylcholine at 37°C for arterioles from control (solid line) and RP/SSc (broken line) subjects. Arterioles were precontracted with norepinephrine (10–6 M). Sample size is 6.

 

    Discussion
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 Abstract
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 Material and methods
 Results
 Discussion
 References
 
Our results indicate that, as in our studies of arterioles isolated from patients with PRP and venules isolated from patients with RP/SSc, increased PTK activity appears to play a role in Raynaud's phenomenon associated with scleroderma [4, 10]. Enhanced PTK activity is consistent with the results of the experiment in which arterioles from control and RP/SSc subjects were cooled from 37° to 31°C in the absence and presence of the protein tyrosine phosphatase inhibitor SOV. In the absence of SOV, arterioles from control subjects dilated slightly when cooled, whereas those from RP/SSc subjects contracted slightly (Figs 2 and 3). Reducing the dephosphorylating action of protein tyrosine phosphatases with SOV did not change the response in arterioles from control subjects but did accentuate the mean effect in arterioles from RP/SSc subjects (Figs 2 and 3). The absence of a change in the response of arterioles from control subjects to cooling in the presence of SOV may occur as a consequence of an increase in PTK activity in endothelial cells from these subjects paralleling that in vascular smooth muscle cells. A number of studies have suggested a role for PTK in the mediation of endothelium-dependent relaxation [11–13]. Thus, in arterioles from control subjects, increased tyrosine phosphorylation in the presence of SOV (Fig. 5) would stimulate both contraction and relaxation with little or no net effect (Fig. 3). Conversely, without a fully functional endothelium the increased tyrosine phosphorylation caused by SOV in arterioles from RP/SSc patients stimulates contraction predominantly (Figs 3 and 6).

The lack of an effect of SOV on the response of arterioles from control subjects to cooling was in contrast to the contraction induced by similar conditions in venules from control subjects [10]. Reduced release of NO and endothelium-derived hyperpolarizing factor from veins compared with arteries may account for the contraction in venules from control subjects [14]. In contrast, as with arterioles from RP/SSc patients, venules from RP/SSc subjects exhibited significant contraction in response to cooling in the presence of SOV [10].

The most pronounced contraction in response to cooling and SOV occurred in an artery from one RP/SSc subject. This artery exhibited a pattern of contraction suggesting vasospasm: gradual contraction followed by rapid and complete collapse of the lumen (Fig. 5). We saw only one artery react in this way to cooling. Perhaps, as in other vascular beds [15], there are differences in contractile properties of skin blood vessels based on location, size or other factors, e.g. involvement in thermoregulation. Although such a highly reactive artery may be uncommon in forearm skin, it may be more common in the skin of the fingers and thus could account for the areas of ischaemia induced by cold in individuals with Raynaud's phenomenon. The reversal of the contractile response by genistein is consistent with the conclusion that increased activity of PTK plays a major role in this response to cooling.

Indeed, when we measured tyrosine phosphorylation using fluorescent immunohistochemistry we observed that increases in fluorescent intensity in arterioles from RP/SSc patients paralleled increases in contraction. Dahdah et al. reported a similar correspondence between contraction and tyrosine phosphorylation in response to cooling in lamb coronary arteries [3].

Whereas the altered PTK activity in arterioles from subjects with primary Raynaud's phenomenon may be genetically determined [16], the enhanced activity of PTK in arterioles from RRP/SSc patients may be related to the elevated levels of a number of growth factors and cytokines in tissues and/or serum from scleroderma patients [17]. Many of these, e.g. platelet-derived growth factor, transforming growth factor ß, IL-6 and IL-1, are known to increase PTK activity in a variety of cell types [18–28].

The similarity of the results of this study to those of a study of isolated arterioles from patients with PRP [4] suggests that the mechanism of vasoconstriction/spasm is similar in the two forms of the phenomenon. In the previous study, as in the present one, we observed contraction in response to cooling in arterioles from patients with PRP but not in those from control subjects. Also, as in this study, contraction of arterioles from PRP patients was associated with increased tyrosine phosphorylation. A recent study of hand–arm vibration syndrome, a form of secondary Raynaud's phenomenon, also supports the possibility that altered signal transduction plays a central role in all forms of Raynaud's phenomenon [29]. The authors determined that fluid oscillation of isolated vascular cells caused an increase in the phosphorylation of the cell signalling component ERK 1/2. Phosphorylation of ERK 1/2 occurs at tyrosine and threonine residues.

The relative unresponsiveness of isolated arterioles from RP/SSc subjects to the relaxant effect of acetylcholine compared with those from control subjects is consistent with reports in the literature of impaired vasodilatory response of RP/SSc subjects to cholinergic agents in vivo [30] and evidence of abnormal endothelial activation in these subjects, i.e. elevated NO synthesis and elevated serum levels of the soluble adhesion molecules E-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 [31, 32]. In a study of isolated skin arterioles from scleroderma subjects, however, Flavahan et al. found no evidence of abnormal responsiveness to acetylcholine [8]. The difference in average duration of the phenomenon between our subject population (13 yr) and that reported by Flavahan et al. (4 yr) may account for the disparate findings. If so, these results would suggest that functionally relevant changes in the endothelium appear later in the progress of the disease than previously thought [33]. Simonini et al. have suggested that hypoxia–reperfusion injury resulting from the vasospasm/relaxation cycle of RP induces the formation of free radicals that damage the endothelium [34]. If endothelial dysfunction is a characteristic of the later stages of the disease, its role in the development of Raynaud's phenomenon, one of the earliest symptoms of the disease, would be obviated. Additionally, endothelial dysfunction is characteristic of a number of vascular diseases, e.g. hypertension, that do not include cold-induced vasospasm among their symptoms. Conversely, patients with PRP do not show evidence of endothelial dysfunction [4, 35].

Unimpaired endothelium-dependent relaxation in individuals with PRP may explain why the severity of the vasospasm they experience is less than that in subjects with RP/SSc [36]. Therefore, although endothelial dysfunction may contribute to the severity of cooling-induced vasospasm in RP/SSc it does not appear to be a primary factor in its occurrence.

In conclusion, as is the case with PRP, the cooling-induced vasospasm of RP/SSc primarily appears to be mediated by the increased activity of PTK. In contrast to PRP, however, RP/SSc may be exacerbated by impaired endothelium-dependent vasodilation.


    Acknowledgments
 
Patient recruitment was greatly facilitated by the cooperation of the Scleroderma Registry funded by the NIH/NIAMS NO1-AR-5-2217. This work was supported by a grant from the NIH (HL-30604).

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Submitted 14 June 2004; revised version accepted 12 November 2004.



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