Published online before print February 9, 2007, doi: 10.1161/01.RES.0000260189.38975.35
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Conditioning Effect of Blood Flow on Resistance Artery Smooth Muscle Myosin Phosphatase
From the Department of Medicine (Cardiology) (H.Z., S.A.F.), Case Western Reserve School of Medicine, Cleveland, Ohio.
Correspondence to Steven A. Fisher, MD, Associate Professor of Medicine and Physiology, Case Western Reserve University School of Medicine, 422 BRB (LC 4958), 2109 Adelbert Road, Cleveland, OH 44106. E-mail saf9{at}po.cwru.edu
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Abstract |
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Myosin phosphatase is the primary effector of smooth muscle relaxation and a target of signaling pathways that regulate vascular tone. The mesenteric small resistance artery and large vessel smooth muscle express distinct isoforms of the myosin phosphatase targeting subunit (MYPT1), and the isoforms in the small resistance artery switch in a disease model of altered blood flow. We thus hypothesized that small resistance artery smooth muscle phenotype is responsive to altered blood flow. To test this hypothesis alternating pairs of rat second order mesenteric arteries were ligated so that the upstream first order mesenteric artery (MA1) is under chronic low flow and the adjacent first order mesenteric artery under chronic high flow. The initial response was similar in high flow and low flow MA1, and included rapid reduction in MYPT1 and switch to the 3' alternative exon skipped/leucine zipper positive MYPT1 isoform. Between 14 to 28 days, MYPT1 abundance was restored along with reversion to the MYPT1 leucine zipper– isoform under chronic high flow. In contrast, under continued low flow, there was further switching to the MYPT1 leucine zipper+ isoform. As would be predicted based on the switch to the MYPT1 leucine zipper+ isoform, the sensitivity for relaxation to the NO donor SIN-1 and to cGMP was increased in the Day28 low flow first order mesenteric artery. We conclude that pulsatile blood flow conditions the phasic program of gene expression in the small resistance artery smooth muscle. The loss of this conditioning effect significantly increases the sensitivity to vasodilator signals in the setting of chronically reduced blood flow.
Key Words: myosin phosphatase • nitric oxide • cGMP • flow • resistance artery
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Introduction |
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We are using myosin phosphatase (MP) as a model for the study of smooth muscle phenotype and its relationship to vascular function in development and disease. MP, by de-phosphorylating myosin, is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate vascular tone. Smooth muscle MP is a hetero-trimer composed of catalytic (PP1c), targeting/regulatory (MYPT1) and 21kDa (M21) subunits. Isoforms of the MYPT1 are generated by alternative splicing of exons in the central and 3' portion of the transcript (reviewed in1). Skipping of the 31 nt exon at the 3' portion of the transcript codes for a subunit that contains a C-terminal leucine zipper (LZ) motif. Inclusion of the 31 nt exon alters the reading frame so that there is no LZ motif and results in a premature stop codon. The expression of the MYPT1 3' alternative exon-included (LZ–) and exon-skipped (LZ+) isoforms is tissue-specific,2–4 developmentally regulated,3,5 evolutionarily conserved,6 and is modulated by disease states.4,7 In the vascular system, the MYPT1 transcript in which the alternative exon is skipped, coding for the LZ+ isoform, predominates in the large vessels that contain smooth muscle of the prototypical tonic (slow) contractile phenotype.3,4 In the portal venous smooth muscle, a prototypical phasic (fast) contractile phenotype, as well as the small resistance artery (SRA) smooth muscle of the mesenteric circulation, the MYPT1 transcript in which the alternative exon is included, coding for the LZ– isoform, predominates.4 The expression of the MYPT1 3' splice variant isoforms are not only markers for the smooth muscle phenotype but also of functional significance. The MYPT1 C-terminal LZ motif is necessary for cGMP-dependent hetero-dimerization of MYPT1 with the cGMP-dependent protein kinase 1
(cGK1) and activation of MP.3,8,9 The activation of MP by NO/cGMP signaling leads to smooth muscle relaxation even at maximally activating concentrations of calcium, ie, calcium desensitization of force production.3,10,11 In a previous study we showed that the phenotype of the mesenteric SRA smooth muscle is not static. In a model of portal hypertension induced by stenosis of the rat portal vein there was a dynamic shift of the mesenteric SRA (and portal vein) smooth muscle MYPT1 to the LZ+ isoform that was part of a generalized switch to a more tonic smooth muscle phenotype.4 Although it has become increasingly clear that the function and phenotype of the SRA smooth muscle differs from that of the large arterial and venous smooth muscle,4,12–16 the mechanisms responsible for the generation of smooth muscle phenotypic diversity in the vascular system, and its modulation in disease, remain for the most part unknown. Given that portal hypertension is a high flow and low vascular resistance state,17 we hypothesized that the changes in MYPT1 isoform expression/smooth muscle phenotype observed in this model could represent a conditioning effect of altered blood flow. To test this hypothesis we implemented the model of DeMey18 and Unthank,19 in which alternating pairs of second order mesenteric arteries are ligated. This results in very low flow (LF) in the upstream first order mesenteric artery (MA1), and a corresponding increase in blood flow (high flow, HF) in the adjacent MA1, which supplies the territory of the occluded vessel through preexisting collateral vessels. Thus there is no confounding tissue ischemia.19 Other MA1s within the same rat remain unligated and thus serve as internal controls. Previous studies have shown that the SRA vessels undergo inward and outward remodeling in this model in response to the chronically reduced and increased flow, respectively.18 Because phenotypic modulation is common to tissue remodeling, we reasoned that the altered blood flow-induced vascular remodeling may be associated with smooth muscle phenotypic modulation. DeMey and coworkers in a previous study using a microarray based approach showed altered expression of a number of smooth muscle specific genes in this model.20 In this study we have focused on the MP and muscle phenotype. We show that SRA MP expression, smooth muscle phenotype, and vascular smooth muscle function are highly responsive to changes in blood flow.
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Materials and Methods |
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For more details, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Animal Model
A 6–0 silk suture ligature was placed to occlude the second order small mesenteric arteries of every other rat MA1.18 Rats were euthanized at fixed intervals 1 to 28 days after surgery.
PCR Analyses of MYPT1 Transcripts
MYPT1, myosin heavy chain (MHC) and myosin light chain (MLC17) splice variants ratios were quantified by RT-PCR as previously described.4 Real-time PCR was performed to measure total transcript abundance of MYPT1 and cyclophilin. Cyclophilin was used as an internal control for normalization, and as previously reported was invariant.20,21
Western Blots
Abundance of MYPT family members and isoforms was examined by Western blot as previously described.4,5 Primary antibodies used were: 1) pan-specific MYPT1 (F38.130, Covance); 2) against the C-terminal LZ sequence present in MYPT family members MYPT1, p85 and M214; 3) myosin light chain kinase (MLCK) (clone K36, Sigma); 4) PP1c 
(Upstate); and 5) smooth muscle -actin (clone 1A4, Sigma).
Vascular Functional Studies
MA1s were mounted on a wire myograph (Model 610 M, Danish Myo Technology, Aarhus, Denmark). The normalization procedure of Mulvany and Halpern was used.22 Dose response of force production to phenylephrine was determined. Vessels were treated with L-NAME to block the production of endogenous NO and then constricted with phenylephrine. Relaxation to cumulative addition of the NO donor SIN-1 was measured. In 2 subsequent sets of experiments: 1) following the incubation with L-NAME and phenylephrine, relaxation to the MLCK inhibitor ML-923 in the presence or absence of 1 µmol/L SIN-1 was measured; and 2) vessels were preincubated with the guanylate cyclase inhibitor ODQ,24 constricted with phenylephrine, then relaxation to cumulative addition of 8Br-cGMP measured.
Statistical Analysis
Data are expressed as mean±SEM. ANOVA and Newman-Keuls test were used for statistical analyses. P<0.05 was considered statistically significant.
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Results |
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MYPT1 Isoform Switching
The smooth muscle of the SRA MA1 expressed predominately the 3' alternative exon-included MYPT1 (82:18 exon-in/exon-out, Figure 1) coding for the C-terminal LZ– isoform, as did the phasic smooth muscle of the portal vein (92:8, Figure 1). The tonic smooth muscle of the aorta expressed predominately the 3' alternative exon-excluded MYPT1 (15:85, Figure 1) coding for the C-terminal LZ+ isoform. Flow velocity was increased by 62% in the HF MA1 and reduced by 67% in the LF MA1 1 day after MA ligature (supplemental Table I of the online data supplement available at http://circres.ahajournals.org.). Twenty-eight days after the ligation, the LF and HF MA1s showed evidence of inward and outward remodeling (supplemental Figure I and Table II), all consistent with a previous study of this model.18 The MYPT1 isoform in the HF MA1 switched from 82:18 to 40:60 within 1 day. This was maintained at 7 to 14 days before increasing to 72:28 at Day 28 (Figure 1A). The initial response to LF was similar, with a change in the ratio at Day 1 and Day 7 to 50:50 (Figure 1B). In contrast to the HF vessels, the MYPT1 ratios shifted further at Day 14 to 29:71 and by Day 28 was 24:76 (Figure 1). The LF MA1 MYPT1 isoform ratio at Day 28 was significantly different from that in the first week, and both were different from control. The HF MA1 MYPT1 isoform ratio in the first week after the change in flow was significantly different from control and the Day 28 value, and the latter 2 were not different from each other (Figure 1C). In these and other assays there were no differences between sham ligated vessels, control MA1s from operated rats, and MA1s from unoperated rats.
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P<0.01 vs control MA1, LF D1–7 MA1 or PV, P>0.05 vs Ao. Ao, aorta; PV, portal vein; MA1, first order mesenteric artery; CTL, control; HF, high flow; LF, low flow; LZ, leucine zipper; Day1, 7, 14, 28, or D1–7, D28 represent days after the change in blood flow.To determine the extent to which the change in MYPT1 isoforms may be indicative of a smooth muscle phenotypic switch, we quantified the relative expression of smooth muscle phenotype-specific MHC-head and MLC17 splice variant transcripts.4,25 In response to HF there was little change in the expression of the MHC-head and MLC17 isoforms (data not shown). In response to chronic LF there was a shift to the expression of the tonic splice variant isoforms of both the MHC-head and MLC17 (Figure 2). In contrast there was no change in the splicing of the MYPT1 central alternative exons in either HF or LF MA1 (supplemental Figure II of the online data supplement available at http://circres.ahajournals.org), demonstrating the specificity in the shifts in the splice variants. The change in the expression of MYPT1, MHC and MLC17 isoforms is consistent with a modulation of the SRA MA1 smooth muscle toward the tonic phenotype under conditions of chronic LF.
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We used an antibody4,5 that specifically recognizes the C-terminal LZ sequence of MYPT family members MYPT1, p85 and M21 to assess the isoforms at the level of the protein. After 28 days of LF the MA1 MYPT1 LZ signal was 3.1±0.7-fold increased over the control MA1 (Figure 3, P<0.01, n=4), consistent with the RT-PCR data showing a switch to the MYPT1 LZ+ isoform. In contrast the MYPT1 LZ signal in the HF MA1 at Day 28 was not different from control MA1 (67±16% of control, n=4, P>0.05), again consistent with the RT-PCR data. Because the absolute MYPT1 LZ signal is confounded by the change in total MYPT1 abundance (see below), an alternative method is to examine the LZ fingerprint in each tissue. In comparing the LZ signal between MYPT1 and p85 and M21, the Day 28 LF MA1 resembles that of the aorta, whereas the Day 28 HF MA1 resembles that of the control MA1 and portal vein (Figure 3), again consistent with the RT-PCR dichotomy of tonic and phasic phenotypes, respectively.
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MYPT1 Abundance
Total MYPT1 protein was downregulated in HF and LF MA1 to 10% or less of control vessels within one day of the change in flow (Figure 4). The reduction in MYPT1 protein was maintained in HF and LF MA1 at Days 7 and 14. At 28 days after the change in flow, MYPT1 protein levels had increased in HF MA1 to 47±7% of control and in LF MA1 to 22±4% of control (n=4 each, P<0.01 versus control and each other). The MP catalytic subunit PP1c was reduced to 64±4% of control MA1 level (HF) and 62±3% (LF) in the first week but did not reach statistical significance (Figure 4, n=4, P>0.05). At Day 28 the HF MA1 expressed 77±2% of control, and the LF MA1 90±12% of control (n=4, P>0.05). This downregulation was not unique to MP as the other major contractile regulatory protein, MLCK, also showed a pattern similar to MYPT1 (Figure 4), being 10% or less of control at Days 1 to 14 in HF and LF MA1, and 50±10% and 26±5% of control in Day 28 HF and LF MA1, respectively (n=4, P<0.05 versus control and each other). These changes were specific, as the abundance of smooth muscle -actin did not change (Figure 4).
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The large decrease in MYPT1 abundance in response to increased or decreased flow could be because of increased protein turnover or reduced synthesis. The rapid reduction in abundance suggests increased catabolism. Pretreatment of the rats with the general proteasomal inhibitor MG132 significantly attenuated the early reduction in MYPT1 abundance of either HF or LF MA1 (Figure 5, percent of control: HF Day 1 plus MG132 70±8%, LF Day 1 plus MG132 68±3%, P<0.01 versus HF Day 1 11±2% or LF Day 1 9±3%; n=3 each). The prolonged reduction in MYPT1 protein abundance was somewhat surprising and suggested reduced synthesis. The level of total MYPT1 transcripts as measured by real-time PCR was significantly reduced from Days 1–14 of both HF and LF MA1 (Figure 6A and 6B). At Day 28 the MYPT1 transcript abundance had increased, with HF MA1 returning to control values (99.8±27.3% of control, n=4, P>0.05 versus control) and LF MA1 increasing to approximately one-half that level (57.5±3.6% of control, n=4, P<0.05 versus control or HF Day 28). The reduction in MYPT1 transcript abundance was specific as there was no change in the abundance of the reference transcript cyclophilin.
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Vascular Function
Given the key role that MP and its isoforms play in the regulation of smooth muscle contractility, we examined contractile properties of the MA1 in the Day 28 LF and HF MA1s. At this point the LF MA1 had switched to the MYPT1 LZ+ isoform and more tonic smooth muscle phenotype, whereas HF MA1 had reverted to the LZ– isoform and more phasic smooth muscle phenotype. The LF Day 28 MA1 was less sensitive to phenylephrine as compared with HF Day 28 MA1 or control MA1 (EC50 of force production LF 4.9±0.1 µmol/L, HF1.5±0.3 µmol/L, control 2.1±0.2 µmol/L) (n=4 to 8, P<0.05 for LF versus control or HF, Figure 7). The maximal force development was significantly different among the 3 groups (P<0.01), but is not normalized for differences in muscle mass.
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The LF Day 28 MA1 was significantly more sensitive to the relaxing effect of SIN-1 as compared with control or HF Day 28 arteries (EC50: LF, n=4, 0.7±0.5 µmol/L versus HF, n=5, 3.2±1.3 µmol/L or control, n=5, 3.9±1.1 µmol/L, P<0.01; Figure 8A). The relaxation of the LF Day 28 MA1 to SIN-1 was not different from a similarly sized vessel containing prototypical tonic smooth muscle, the 3-week-old mouse aorta (n=4, EC50: 0.2±0.1 µmol/L, P>0.05). SIN-1 nearly completely relaxed all vessels at the highest concentration. The relaxation of all vessels to 100 µmol/L SIN-1 was abolished by pretreatment with the guanylyl cyclase inhibitor ODQ (1 µmol/L) (data not shown), indicating that the response to SIN-1 required the generation of cGMP.
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Because the difference in response to the NO donor could reflect differences in the generation of cGMP, relaxation responses to the nonhydrolyzable cell permeable analogue 8Br-cGMP were measured. The dose-response to the cGMP analogue showed a similar increase in sensitivity in the LF MA1, whereas HF and control vessels were not different (EC50: LF, n=5, 9.0±1.9 µmol/L versus HF, n=5, 46.3±17.4 µmol/L or control n=4, 38.5±4.2 µmol/L, P<0.01; Figure 8B). The response of the LF MA1 was similar to that previously reported for the aorta.3,26
Because vascular smooth muscle force is primarily determined by the opposing activities of MP and MLCK in determining the level of myosin light chain phosphorylation, we wanted to isolate the effect of NO signaling to the MP. We therefore tested the response to the NO donor SIN-1 in the presence of the inhibitor of MLCK (ML-9, 25 µmol/L). Control, HF Day 28 and LF Day 28 MA1s completely relaxed to ML-9 alone at similar rates (n=4 each, Figure 8C). Addition of 1 µmol/L SIN-1 to ML-9 significantly increased the rate of relaxation in LF Day 28 MA1 but not in the HF Day 28 and control MA1 (Figure 8C, P<0.05, n=3 each), providing evidence for selective activation of MP by 1 µmol/L SIN-1 in the LF Day 28 MA1.
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Discussion |
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In this study we have examined how alterations in blood flow in the micro-circulation affect the expression of the smooth muscle MP, a key target of signaling pathways that regulate vascular tone. The early response of the MA1 to either increased or decreased blood flow, measured at 24 hours after the ligation of the second order MAs, included a reduction in the abundance of MP protein and mRNA and a shift toward the 3' exon-skipped splice variant of MYPT1 that codes for the LZ positive isoform. The reduction in MP protein abundance was not unique, as the other major contractile regulatory protein, MLCK, changed with a similar time course. It was specific, as there was no change in the abundance of the myofibrillar protein smooth muscle
-actin. The near complete (90%) disappearance of MYPT1 and MLCK protein within 24 hours of the change in flow, and the ability to largely prevent its disappearance with the proteasomal inhibitor MG132, suggests that the protein is degraded because of activation of the proteasome by the altered flow. This would be consistent with other studies showing that smooth muscle cells activate the proteasome when stimulated to proliferate in vitro,27 and that regulatory proteins are more susceptible to proteasomal degradation than are actin and myosin.28 The MYPT1 mRNA levels are also significantly and specifically reduced, as previously suggested by a micro-array study.20 This would suggest suppressed synthesis of MYPT1, which in combination with MYPT1 degradation likely underlies the prolonged reductions in MYPT1 protein abundance in the HF and LF MA1s. Further studies are required to test the role of proteasomal activation in flow-induced SRA remodeling. In addition to changes in MYPT1 abundance there are also significant MYPT1 isoform shifts in this model of altered flow. Under both high and low flow conditions there is a shift toward the exon-excluded isoform within 24 hours. Currently little is known about the factors that control the splicing of MYPT1 or other alternative exons during smooth muscle phenotypic modulation. The proximate stimulus for the early change in SRA smooth muscle MYPT1 3' splice variants, or for that matter the other changes in gene expression discussed above, are not known. That similar changes occur at the early time points in both the high and low flow MA1, but not adjacent control MA1s, suggests a signal that is generated in response to a change in blood flow, be it positive or negative, as has been demonstrated for reactive oxygen species (ROS) generation in endothelial cells.29 Because pressure and flow are inter-dependent variables, it is also possible that altered tensile stress, or an interaction of flow and pressure, is the trigger. Somewhat unique to this distal ligation model is that the upstream artery is exposed to systemic pressure but with very low blood flow. However, arguing against a primary role for altered tensile stress are: 1) similar phenotypic changes are observed when the MA1 is ligated proximally (data not shown), reducing pressure and flow beyond the ligature to very low levels: and 2) phenotypic switching is not observed in the MA1 in a model of systemic hypertension (manuscript in preparation). In vitro experiments may be able to isolate the effects of flow (shear) and pressure as independent variables influencing SRA smooth muscle phenotype.
At later time points the responses of the HF and LF vessels diverge. In the HF vessels by 28 days the abundance of MYPT1 mRNA and protein have increased and the 3' splice variants have returned to predominance of exon-inclusion coding for the isoform that lacks the C-terminal LZ motif. The reason for return to the control phenotype likely reflects the abatement of the inciting stimulus because of the outward remodeling of the artery (18 and this study). In contrast under continued LF from 14 to 28 days there is even further switching to the MYPT1 3' exon-skipped splice variants coding for the C-terminal LZ motif. This switching to the MYPT1 LZ+ isoform, along with the approximately 50% reduction in MYPT1 and MLCK abundance, and shifts in myosin heavy and light chain splice variant isoforms, are indicative of a modulation to the tonic smooth muscle phenotype. Given that the HF MA1 reverts to the control smooth muscle phenotype with a significant phasic component of gene expression, while the LF vessel continues to modulate to the tonic smooth muscle phenotype, we propose that the pulsatile blood flow has a conditioning effect on the SRA smooth muscle that is required for the phasic program of gene expression.
We selected the Day 28 vessels to study the functional significance of the isoform switching as MYPT1 protein abundance had rebounded but the isoform profiles were distinct in the LF versus HF and control vessels. We have previously shown in two different developmental models, the rat portal vein5 and the chicken gizzard,3 that the switch from MYPT1 LZ positive to negative isoforms as part of the acquisition of the phasic phenotype is associated with the development of resistance to cGMP-mediated calcium desensitization of force production. In the current study the expression of the MYPT1 LZ+ isoform in the Day 28 LF MA1 is also associated with increased sensitivity to NO donor and cGMP-mediated relaxation. This result is consistent with the paradigm that has emerged from a number of in vitro and in vivo studies indicating that the C-terminal LZ motif of MYPT1 is required for the activation of MP by the cGMP-dependent protein kinase leading to calcium desensitization of the myofilaments.3,8,9 The mechanism by which cGK1 enhances MP activity remains undefined, thus there is currently no biochemical marker for MP activation. In this study we have used a standard surrogate marker, the ability of NO/cGMP signaling to enhance relaxation with MLCK pharmacologically blocked with ML-9. Because the rate of smooth muscle relaxation reflects the rate of dephosphorylation of myosin, differences in the rate of relaxation between tissues when MLCK activity is blocked reflect differences in the activity of MP.
Control, HF and LF MA1s all nearly completely relaxed at the highest concentration of the NO donor SIN-1, 100 µmol/L. However the LF MA1 was significantly more sensitive to the relaxing effect of SIN-1, with an EC50 that was 
4.5-fold lower than that of the HF and control MA1, which did not differ. The sensitivity of the LF MA1 to SIN-1 was not different from that of the tonic aorta. This data are consistent with prior studies showing that NO plays less of a role in endothelium-derived relaxations of the SRAs as compared with the large artery smooth muscle.14,15,30 In the current study we have shown that the contractile properties and phenotype of the mesenteric SRA smooth muscle are plastic, with modulation to the tonic smooth muscle phenotype with similar NO donor sensitivity under chronic LF conditions. It remains to be determined how the phenotypic switch affects the regulation of blood flow and tissue perfusion under chronic low flow conditions in vivo. Our data would suggest that the loss of the phasic characteristics of the SRA smooth muscle and switch to the tonic /MYPT1 LZ+ phenotype would allow for maximal dilation to NO/cGMP signaling under conditions of chronic low flow.
One limitation of this study is that we measured changes in force under isometric conditions in a wire myograph system after -adrenergic induced force development. A prior study using a pressurized myograph system measured changes in vessel diameter in vitro and observed a reduction in the sensitivity of the LF MA1 to acetylcholine or the NO donor sodium nitroprusside, but an increased response to flow-mediated dilation.31 Differences besides the experimental apparatus in the 2 studies include: 1) the use of vasopressin to activate force before sodium nitroprusside exposure in the study by Pourageaud and DeMey, which produced more force than the -adrenergic agonist norepinephrine. It is possible that different pathways for force activation also have different susceptibilities to inhibition by NO/cGMP signaling; 2) preincubation with L-NAME in our experiments to block the effect of endogenous NO; and 3) the use of different NO donor drugs, each of which may have effects independent of the generation of cGMP (reviewed in32). We believe that force measurements are a better indicator of the state of muscle contraction than are changes in vessel diameter, since a linear relationship between force in smooth muscle and level of myosin phosphorylation has been established.33 On the other hand, measurements of changes in vessel diameter under conditions of pressure and flow are more physiologically relevant. Our study also shows increased sensitivity of the LF MA1 to cGMP, consistent with the increased sensitivity to the NO donor signaling through cGMP.
In conclusion, this study establishes that the SRA smooth muscle phenotype and sensitivity to NO/cGMP signaling is plastic and highly influenced by chronic changes in blood flow. In addition to MP a number of other targets of NO/cGMP signaling have been established or suggested that regulate smooth muscle calcium flux and calcium sensitivity of force production.34 Understanding how the expression and function of these targets is altered under conditions of chronically altered blood flow will have important implications for the function of vasodilators in the many diseases in which blood flow and tissue perfusion are perturbed.
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Acknowledgments |
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Source of Funding
This study was supported by NIH grant HL66171 (to S.A.F.).
Disclosures
None.
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Footnotes |
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Original received October 7, 2006; revision received January 22, 2007; accepted January 25, 2007.
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References |
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