Published online before print March 13, 2008, doi: 10.1161/CIRCRESAHA.107.169896
© 2008 American Heart Association, Inc.
Molecular Medicine |
Targeted Deletion of PTEN in Smooth Muscle Cells Results in Vascular Remodeling and Recruitment of Progenitor Cells Through Induction of Stromal Cell–Derived Factor-1
From the Department of Medicine, Divisions of Renal Diseases and Hypertension (R.A.N., P.A.S., S.B.F., N.K.-A., J. Cooper, M.C.M.W.-E.), Pulmonary Sciences and Critical Care Medicine (J. Crossno), Cardiovascular and Pulmonary Research (R.A.N., J. Crossno, P.J.G., M.C.M.W.-E.), University of Colorado Denver; and Veterans Affairs Medical Center (J. Crossno), Denver.
Correspondence to Mary C.M. Weiser-Evans, Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Denver, Denver, CO 80262. E-mail mary.weiser{at}uchsc.edu
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Abstract |
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We previously showed that changes in vascular smooth muscle cell (SMC) PTEN/Akt signaling following vascular injury are associated with increased SMC proliferation and neointima formation. In this report, we used a genetic model to deplete PTEN specifically in SMCs by crossing PTENLoxP/LoxP mice to mice expressing Cre recombinase under the control of the SM22
promoter. PTEN was downregulated with increases in phosphorylated Akt in major vessels, hearts, and lungs of mutant mice. SMC PTEN depletion promoted widespread medial SMC hyperplasia, vascular remodeling, and histopathology consistent with pulmonary hypertension. Increased vascular deposition of the chemokine stromal cell–derived factor (SDF)-1 and medial and intimal cells coexpressing SM--actin and CXCR4, the SDF-1 receptor, was detected in SMC PTEN-depleted mice. PTEN deficiency in cultured aortic SMCs induced autocrine growth through increased production of SDF-1. Blocking SDF-1 attenuated autocrine growth and blocked growth of control SMCs induced by conditioned media from PTEN-deficient SMCs. In addition, SMC PTEN deficiency enhanced progenitor cell migration toward SMCs through increased SDF-1 production. SDF-1 production by other cell types is regulated by the transcription factor hypoxia-inducible factor (HIF)-1. We found SMC nuclear HIF-1 expression in PTEN-depleted mice and increased nuclear HIF-1 in PTEN-deficient SMCs. Small interfering RNA–mediated downregulation of HIF-1 reversed SDF-1 induction by PTEN depletion and inhibition of phosphatidylinositol 3-kinase signaling blocked HIF-1 and SDF-1 upregulation induced by PTEN depletion. Our data show that SMC PTEN inactivation establishes an autocrine growth loop and increases progenitor cell recruitment through a HIF-1–mediated SDF-1/CXCR4 axis, thus identifying PTEN as a target for the inhibition of pathological vascular remodeling.
Key Words: smooth muscle cell • PTEN • neointima • autocrine growth • conditional knockout mouse
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Introduction |
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Smooth muscle cell (SMC) accumulation in the arterial intima is a key event in the pathogenesis of atherosclerosis, postangioplasty/in-stent restenosis, and graft arteriosclerosis,1 with changes in the biological function and phenotype of SMCs contributing to the pathology.2 These conditions are characterized, to varying degrees, by dedifferentiation, migration, and proliferation of medial-derived SMCs to form the neointima. Recent data suggest that bone marrow–derived, circulating, and/or resident progenitor/proinflammatory cells are recruited to the injured vessel, differentiate down a SMC lineage, and proliferate, thereby contributing to neointimal lesion formation.3–6 Compelling evidence supports the contribution of both processes to intimal hyperplasia, and major advances have identified numerous factors involved in this complex pathobiology. However, the underlying mechanism(s) initiating lesion formation are not clearly defined. Increased SMC production of chemokines, such as stromal cell–derived factor (SDF)-1
(CXCL12), has been shown to be centrally involved in progenitor cell recruitment,7 although their role in inducing an autocrine growth pathway within the artery wall itself is unknown. We focused on the hypothesis that SMCs are central mediators of the injury response. Perturbations in SMC signaling, result in the production of soluble factors that regulate significant SMC hyperplasia and progenitor/proinflammatory cell recruitment through an autocrine/paracrine mechanism. Under physiological conditions, the mature blood vessel is a highly quiescent tissue,8,9 suggesting that pathological vascular remodeling requires the inactivation of active growth inhibitory pathways before rendering SMCs permissive to growth stimulation. Several studies support the concept that neointimal SMCs exhibit a distinct growth phenotype similar to fetal-derived SMCs that is characterized by mitogen-independent proliferation and, despite the expression of functional growth factor receptors (eg, platelet-derived growth factor [PDGF] receptor-β), a blunted mitogenic response to exogenous growth factors known to stimulate medial-derived SMCs.10–12 Our previous studies demonstrated that mitogen-independent growth is actively repressed in differentiated SMCs,12 suggesting that loss of growth suppressors likely leads to enhanced SMC growth potential in pathological conditions such as restenosis. Our subsequent work strongly implicated the tumor suppressor PTEN as a potent, endogenously produced inhibitor of mitogen-independent SMC proliferation.13,14
PTEN is a dual-specificity lipid and protein phosphatase that inhibits cell proliferation, survival, and growth predominantly through dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, thus antagonizing phosphatidylinositol 3 (PI3)-kinase–mediated signaling events.15–17 Regulation of the PI3-kinase/Akt/mTOR signaling pathway plays a pivotal role in SMC proliferation in culture and during the pathogenesis of restenosis.14,18–20 Important clinically, the use of rapamycin-eluting stents to inhibit mTOR signaling has significantly reduced the incidence of in-stent restenosis.21,22 Previous studies demonstrated that PTEN overexpression reduces intimal hyperplasia in rat carotid artery injury and saphenous vein graft models,19,23 and upregulation of SMC-derived PTEN attenuates atherosclerotic lesion formation in high-fat-fed rabbits, thus implicating PTEN as an antiatherogenic protein.24 Our studies show that temporally controlled PTEN activity correlated with significant alterations in SMC growth rate during vascular development and after experimental vascular injury.13,14 Importantly, inactivation of PTEN in the setting of vascular injury, leading to constitutive Akt activation, is an early and critical event involved in neointima formation. The molecular events mediating the effects of PTEN inactivation on SMC hyperplasia and on progenitor cell recruitment, however, have yet to be identified. To define the role of PTEN signaling in this context, we examined the effects of conditional deletion of SMC PTEN.
By mating PTENLoxP/LoxP mice to transgenic mice expressing Cre recombinase under the control of the SM22 promoter, we show here that SMC-specific PTEN mutant mice (PTEN knockout [KO]) exhibit many features associated with pathological vascular remodeling, including remarkable medial and intimal SMC hyperplasia as well as vascular recruitment of progenitor cells. In particular, PTEN depletion results in hypoxia-inducible factor (HIF)-1–mediated production of the chemokine SDF-1, which induces an autocrine SMC growth loop and increases progenitor cell migration through a paracrine signaling mechanism. Our data thus suggest that an alteration in SMC PTEN signaling serves as one of the initiating determinants driving pathological vascular remodeling.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animals and Generation of PTEN mutant mice
SMC-specific PTEN-null mutant mice were generated by crossing SM22-Cre transgenic mice to PTENflox/flox mice. PTENflox/flox;SM22-Cre/+; R26R/+ mice were generated by interbreeding PTENflox/flox mice with R26R mice followed by crossing PTENflox/+;SM22-Cre/+ to PTENflox/flox; R26R/+ mice. Animals were bred and maintained following guidelines approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver Health Sciences.
Additional Methods
An expanded Materials and Methods section containing details regarding animals and generation of PTEN mutant mice; cell culture; generation of stable short hairpin (sh)RNA-expressing SMCs and small interfering (si)RNA transfections; quantitative RT-PCR, Western blot analysis, and ELISA; immunohistochemistry, immunofluorescence, LacZ staining; peripheral blood mononuclear cell (PBMC) isolation, flow cytometry, and cell labeling; growth and transmigration assays; and statistical analysis is available in the online data supplement.
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Results |
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Smooth Muscle–Specific PTEN Depletion Promotes Spontaneous Development of Arterial Remodeling and Histopathology Consistent With Pulmonary Hypertension
Using a rat angioplasty model, we established that PTEN inactivation was localized to the developing neointima and exclusively in replicating neointimal SMCs13,14 strongly implicating inactivation of PTEN in the pathogenesis of injury-induced intimal hyperplasia. Therefore, to define the role of PTEN specifically in smooth muscle (SM) tissues using a genetic model, PTENLoxP/LoxP mice25 were mated to transgenic mice expressing Cre recombinase (Cre) under the control of the SM22
promoter (Figure IA in the online data supplement and Materials and Methods).26 Cre specificity was verified by crossing SM22-Cre transgenic mice to ROSA26 Reporter (R26R) mice, as described previously (supplemental Figure IB).26 Crossing SM22-Cre mice with PTENflox/flox mice generated control mice (PTENflox/flox; +/+, PTENflox/+; +/+, and PTENflox/+; Cre/+) and homozygous mutant mice (PTENflox/flox; Cre/+; PTEN KO). Normal Mendelian ratios of all genotypes were observed in embryos and at birth (data not shown). PTEN KO mice, however, were smaller (supplemental Figure ID) and weaker, and the majority died by 3 weeks of age (all before 6 weeks); no differences among the other 3 groups were observed. All studies reported hereafter compared 20-day-old PTEN KO mice with PTENflox/flox; +/+ control littermates (WT). PCR for the PTEN
allele (indicating Cre activity) in DNA extracted from aortic, carotid artery, heart, and lung tissues revealed the expected PCR product in PTEN KO but not WT mice (supplemental Figure IC).27 Western blotting showed significant reductions in total PTEN protein in large arteries, hearts, and lungs of PTEN KO mice compared with WT, with an accompanying increase in phospho-Akt levels, demonstrating a functional loss of PTEN activity (supplemental Figure ID).
PTEN KO mice exhibited significant SMC hyperplasia in major vessels, leading to increased medial thickness along with aortic intimal accumulation of SM--actin–positive cells (Figure 1A). Increased numbers of SM--actin–positive, 5-bromodeoxyuridine (BrdUrd)-positive replicating cells were observed throughout the aortic and pulmonary arterial walls in PTEN KO mice (Figure 1B). In addition, PTEN KO mice spontaneously developed right ventricular hypertrophy and pulmonary vascular remodeling at Denver, Colo, ambient air (1609 meters). RV/LV+S ratios, a measure of right ventricular hypertrophy, were significantly higher in PTEN KO compared with WT mice, suggesting the development of pulmonary hypertension (Figure 1C). Compared with WT, PTEN KO mice exhibited increased wall thickness of small pulmonary arteries, occluded precapillary arterioles, and reduced alveolarization (Figure 1D). Occluded precapillary vessels stained positive for von Willebrand factor (an endothelial marker) and SM--actin, characteristic of plexiform lesions observed in human primary pulmonary hypertension.28 Increased numbers of replicating SM--actin–positive and –negative cells were observed throughout the lungs of PTEN KO mice, including small, muscularized pulmonary arteries (Figure 1E). In addition, increased numbers of replicating coronary artery SMCs, coronary perivascular cells, cardiomyocytes, and interstitial fibroblasts were observed in hearts of PTEN KO mice (supplemental Figure II). Cardiomyocyte proliferation was consistent with early developmental cardiac expression of SM22 and, therefore, inactivation of PTEN. Enhanced extracellular matrix deposition was detected in major arteries and in perivascular locations in lungs and coronary vessels of mutants (supplemental Figure III), demonstrating vascular, lung, and cardiac interstitial fibrotic changes. The observed myocardial fibrosis is a key pathological feature of heart failure, the likely cause of death of mutants.
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Recruitment of Circulating Progenitor Cells and Increased SDF-1 Expression in PTEN KO Mice
In addition to the above, PTEN KO mice consistently exhibited significant splenomegaly (supplemental Figure IVA). Increased bone marrow hyperplasia and hyperplasia of splenic red pulp, consistent with increased extramedullary hematopoiesis, were detected in PTEN KO mice (supplemental Figure IVB through IVD); no changes were observed in livers or kidneys (not shown). Because enhanced bone marrow progenitor/hematopoietic cell mobilization can result in extramedullary hematopoiesis, we hypothesized that SMC-specific PTEN deletion results in production of systemic factors involved in mobilization and trafficking of bone marrow–derived progenitors. To determine whether histological changes were accompanied by increases in peripheral blood progenitor cells, PBMCs were analyzed by 2-color flow cytometry for the hematopoietic leukocyte marker CD45; the progenitor markers CD34, c-Kit, Sca-1, and CXCR4; the endothelial cell progenitor marker FLK-1; or the monocyte progenitor marker CD14. Higher numbers of circulating CD34+, c-Kit+, CD14+, and CXCR4+ hematopoietic cells were detected in PTEN KO mice compared with WT (Figure 2A); no changes in FLK-1+ or Sca-1+ cells were detected (supplemental Figure VA). In PTEN KO mice expressing the ROSA26 reporter allele, where LacZ is expressed in cells lacking PTEN, no Cre activity was detected in bone marrow, therefore direct inactivation of PTEN in bone marrow cells did not mediate progenitor cell mobilization (supplemental Figure IVE).
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To determine whether mutant-derived PBMCs traffic to the vasculature, PBMCs isolated from 3-week-old WT or PTEN KO donor mice were labeled with fluorescent quantum dot nanocrystals, and reinjected retroorbitally to either 3-week-old WT or PTEN KO recipient mice. Target tissues were analyzed for fluorescence 24-hour postinjection; cells were scored positive if they contained significant amounts of cytoplasmic, granular fluorescent nanocrystals plus a nucleus. No labeled cells were detected on the aorta of control animals (WT donor-to-WT recipient; WT donor-to-KO recipient; KO donor-to-WT recipient). Only single positive cells were detected in the lungs (Figure 2B), and these were not associated with recipient lung alveolar or vascular surfaces. In contrast, labeled cells were identified on aortic intimal and adventitial surfaces and in lungs of KO donor-to-KO recipient mice; large numbers of labeled progenitors were identified in the lungs (Figure 2B). SM--actin–positive and –negative PBMCs were detected often in a perivascular location (Figure 2C), suggesting some of the recruited PBMCs differentiate toward SMCs. Increased numbers of labeled cells were also found in spleens (supplemental Figure VIA), but not livers (not shown), of KO donor-to-KO recipient mice. Consistent with increased trafficking of PBMCs, accumulation of CD45+ and c-Kit+ cells was observed on major arteries and in perivascular locations in lungs of PTEN KO mice (Figure 2C and supplemental Figure VB). Taken together, these results support the concept that SMC-specific PTEN depletion produces a local microenvironment favorable for trafficking and accumulation of circulating progenitor cells.
Recent data support a role for the chemokine SDF-1 and its receptor expressed by hematopoietic progenitor cells, CXCR4, in promoting vascular remodeling.29–31 Changes in the SDF-1 gradient from bone marrow to blood result in movement of precursor cells into the circulation.7 Increased plasma levels and local vascular accumulation of SDF-1 in response to vascular injury recruit CXCR4+ bone marrow cells to remodeling vessels.7,32,33 Because circulating CXCR4+ PBMCs were detected in PTEN KO mice, SDF-1 expression in tissues from WT and PTEN KO mice was examined. There was a nonsignificant trend toward increased serum SDF-1 levels (Figure 3A) and increased vascular and perivascular deposition of SDF-1 in aortae and lungs of PTEN KO mice (Figure 3B). This was associated with intimal accumulation of CXCR4+ cells in the vasculature (Figure 3C) and in spleens of PTEN KO mice (supplemental Figure VIB). CXCR4 expression was also detected on medial SMCs of WT and KO mice (Figure 3C), suggesting SMCs express functional SDF-1 receptors. These results support a model in which SMC-specific PTEN inactivation indirectly promotes vascular recruitment of progenitor cells through induction of SDF-1. Because CXCR4 was detected on medial SMCs, increased SMC-derived SDF-1 could also directly affect the biological function of SMCs.
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PTEN Depletion Induces SMC Autocrine Growth and Enhances Progenitor Cell Migration Through Increased SDF-1 Production
To verify the direct effect of PTEN depletion on SMC function in vitro, aortic SMCs stably expressing PTEN-specific shRNA were generated. PTEN depletion resulted in enhanced Akt activity under basal conditions and in response to PDGF stimulation compared with SMCs transfected with empty vector (CTRL). Increased Akt phosphorylation was blocked by treatment with LY294002 to inhibit PI3-kinase activity, demonstrating a functional loss of PTEN (Figure 4A). Primary aortic SMCs transfected with control scrambled or PTEN-specific siRNA exhibited similar responses (supplemental Figure VII). PTEN depletion increased cell proliferation under basal conditions and reduced responsiveness to PDGF-BB, a known mitogen for medial-derived SMCs (Figure 4B), similar to what is observed in fetal and neointimal SMCs.10–12 As with fetal and neointimal SMCs, reduced mitogenic responsiveness to PDGF was not attributable to a loss of functional PDGF receptors (data not shown). Conditioned media from PTEN-depleted SMCs stimulated CTRL SMC proliferation, indicating that PTEN depletion results in secretion of a mitogen, which may act in an autocrine fashion (Figure 4C). Consistent with results in PTEN KO mice, PTEN deficiency in vitro increased SDF-1 mRNA and secretion into the cell media (Figure 4D). Recombinant SDF-1 stimulated proliferation of CTRL SMCs that was blocked with an anti–SDF-1 neutralizing antibody (Figure 4E). A neutralizing anti–SDF-1 antibody also attenuated autocrine growth of PTEN-depleted SMCs and blocked growth of CTRL SMCs induced by conditioned media from PTEN-depleted SMCs (Figure 4E). This effect was specific for SDF-1, because neutralization of PDGF had no effect on growth responses to conditioned media from PTEN-depleted SMCs (Figure 4E). Consistent with in vivo data, CXCR4 was expressed by CTRL and PTEN-depleted SMCs (Figure 4E, inset), thus supporting the concept that SDF-1 upregulation vis a vis PTEN loss induces an autocrine growth loop involving an SDF-1/CXCR4 axis.
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To determine whether increased production of SDF-1 by PTEN-deficient SMCs results in enhanced migration of circulating progenitor cells in vitro, CTRL and PTEN-depleted SMCs were cocultured with PBMCs from WT or PTEN KO mice using a Transwell system (Figure 5A). PBMCs from both WT and PTEN KO mice showed increased migration toward PTEN-depleted SMCs compared with CTRL SMCs, consistent with enhanced SDF-1 levels mediating chemotaxis of CXCR4+ progenitor cells (Figure 5B). Higher numbers of migrating PBMCs were observed from PTEN KO mice compared with WT mice, consistent with higher numbers of circulating CXCR4+ cells in PTEN KO mice. Migration toward PTEN-depleted SMCs was attenuated by the addition of neutralizing anti–SDF-1 and increased toward CTRL SMCs in response to recombinant SDF-1 (Figure 5B), suggesting that enhanced secretion of SDF-1 induced by PTEN depletion drives progenitor cell migration through a paracrine signaling mechanism.
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Vascular injury–induced SDF-1 expression in SMCs was recently shown to be regulated by the transcription factor HIF-1 in a hypoxia-independent manner,34 and previous studies showed HIF-1 upregulation in other cell systems is mediated by PI3-kinase/Akt signaling.35 We thus examined the expression of HIF-1 in PTEN KO mice and SMCs. Compared with WT mice, strong, nuclear HIF-1 staining was observed in aortic SMCs of PTEN KO mice (Figure 6A). Consistent with in vivo results, PTEN-depleted SMCs expressed increased HIF-1 mRNA and HIF-1 protein in nuclear protein extracts compared with CTRL SMCs (Figure 6B and 6C). siRNA-mediated depletion of HIF-1 reversed SDF-1 induction by PTEN depletion (Figure 6D), and inhibition of PI3-kinase signaling blocked HIF-1 and SDF-1 upregulation induced by PTEN depletion (Figure 6E), suggesting that SMC PTEN inactivation directly increases HIF-1 expression, leading to SDF-1 upregulation.
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Discussion |
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We previously demonstrated that vascular injury leads to inactivation of PTEN in SMCs.13,14 The goal of the present study was to examine the consequences of PTEN inactivation using a genetic model in which PTEN is specifically deleted in SMCs. Because changes in SMC PTEN signaling are observed in the early stages following vascular injury, we postulated that PTEN loss would initiate key events involved in pathological vascular remodeling. Our data indicate that deletion of PTEN is sufficient to induce proliferation of medial SMCs and accumulation of SMCs in the vascular intima. Our findings are consistent with a previous study that used a similar genetic inactivation strategy and showed SMC PTEN loss resulted in widespread SMC hyperplasia associated with the initiation of leiomyosarcoma development by 2 months of age.36 We did not detect leiomyosarcomas because our mutants rarely survived past 21 days, considerably shorter than what was reported in this study (mean, 70.3 days). Differences are likely attributable to genetic background, because our mice were fully backcrossed to the C57BL/6 background. Nevertheless, in both instances, PTEN depletion specifically in SMCs induced significant SMC hyperplasia in the absence of other stimuli. In addition, we report that SMC PTEN depletion is sufficient to recruit hematopoietic progenitor cells to the vasculature. Based on both our in vivo and in vitro data, a critical mediator of PTEN inactivation is increased HIF-1
–mediated production of SDF-1 by SMCs.
Several studies have demonstrated that vascular remodeling is associated with increased chemokine production by SMCs, as well as other cell types.7,37 Increased SMC production of SDF-1 has been implicated in the recruitment of bone marrow–derived progenitor cells expressing the SDF-1 receptor CXCR4, which contribute to neointima formation. Vascular accumulation of SDF-1 is critical for targeting CXCR4-positive cells to the site of injury. Consistent with these studies, our data show that in PTEN KO mice, CXCR4-expressing intimal cells and PBMCs homing to the vasculature express SM--actin, suggesting recruitment of SMC progenitors. The role of SDF-1 on SMC proliferation, however, has been less studied. Consistent with previous studies,38 our data indicate that SMCs express CXCR4, and therefore production and release of SDF-1 would be anticipated to establish an autocrine growth loop. Our in vitro studies demonstrate that PTEN silencing is sufficient to induce SDF-1 expression and increase autonomous growth of SMCs. In fact, blocking SDF-1 reduced autonomous proliferation of PTEN-depleted cells to control levels, suggesting that, in addition to its effects on progenitor cell recruitment, increased SMC-derived SDF-1 may be a major regulator of enhanced SMC proliferation.
Although we have not defined the downstream effectors leading to SDF-1 induction, our data suggest that Akt-dependent upregulation of HIF-1 is likely to be important. Hypoxia-induced SDF-1 upregulation in endothelial cells was shown to be HIF-1–dependent,39 and forced overexpression of PTEN in glioma cells was shown to significantly reduce HIF-1 expression.40 Karshovska et al34 demonstrated induction of HIF-1 in SMCs following vascular injury; HIF-1 inhibition resulted in reduced neointimal area and decreased SDF-1 expression. Our studies show that inactivation of PTEN in SMCs is sufficient to upregulate HIF-1 in an Akt-dependent manner; blocking HIF-1 upregulation reverses the induction of SDF-1. Studies are ongoing to determine the molecular pathways responsible for the regulation of HIF-1 and SDF-1 production mediated by PTEN inactivation.
In summary, we report that SMC PTEN-deficient mice spontaneously develop features in both the systemic and pulmonary vasculature associated with pathological vascular remodeling that are mediated, at least in part, through the induction of the chemokine, SDF-1. It should be noted that, although SDF-1/CXCR4 signaling has been shown to promote pathological vascular remodeling, recent data also suggest SDF-1/CXCR4 signaling exerts protective effects in primary atherosclerosis.41 Unfortunately, early lethality in the present model precludes the ability to fully examine molecular events associated with the pathogenesis of remodeling on PTEN inactivation. Although our data suggest PTEN inactivation promotes medial SMC proliferation and the potential recruitment of SMC progenitors, these mice cannot be used as recipients for bone marrow transplant studies. It is, therefore, difficult to determine whether recruited cells are pathological or protective in the present system. We are in the process of developing an inducible SMC-specific system to more clearly answer these questions. Nevertheless, our data are consistent with the proposal that sustained SMC PTEN signaling serves as a central regulator of the vascular injury response and, therefore, have important clinical implications in pathologies associated with vascular remodeling, including restenosis and pulmonary hypertension.
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Acknowledgments |
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Sources of Funding
Supported by NIH grants EB003999 (to M.C.M.W.-E.) and DK19928 (to R.A.N.).
Disclosures
None.
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Footnotes |
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Original received December 13, 2007; revision received March 3, 2008; accepted March 5, 2008.
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References |
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