Sca-1+ Progenitors Derived From Embryonic Stem Cells Differentiate Into Endothelial Cells Capable of Vascular Repair After Arterial Injury
Background— Embryonic stem cells possess the ability to differentiate into endothelium. The ability to produce large volumes of endothelium from embryonic stem cells could provide a potential therapeutic modality for vascular injury. We describe an approach that selects endothelial cells using magnetic beads that may be used therapeutically to treat arterial injury.
Methods and Results— Large numbers of endothelial cells (ECs) with high purity were produced using Sca-1+ cells isolated with magnetic beads from predifferentiated embryonic stem cells (ESCs) cultured in α-MEM containing 10 ng/mL VEGF165 for a minimum of 21 days (esEC). The transcription regulator histone deacetylase (HDAC3) was essential for VEGF-induced EC differentiation. Immunofluorescence or fluorescence-activated cell sorter (FACS) analysis revealed that esECs expressed a full range of EC lineage-specific markers including CD31, CD106, CD144, Flk-1, Flt-1, and von Willebrand factor (vWF). FACS analysis confirmed that 99% of esECs were CD31-positive and 75% vWF-positive. Furthermore, almost all cells were positive for DiI-acLDL uptake. When matrigel containing esECs was subcutaneously implanted into mice, various vessel-like structures were observed indicating their endothelial cell like phenotype. In keeping with this, when esECs infected with adenovirus-LacZ were injected into denuded femoral arteries of mice, they were found to form a neo-endothelium that covered the injured areas (86%±13.6%), which resulted in a 73% decrease in neointimal area 2 weeks after injury.
Conclusions— We conclude that Sca-1+ cells can differentiate into functional ECs via activation of HDAC3, accelerating re-endothelialization of injured arteries and reducing neointima formation.
Balloon angioplasty and stenting is routinely used in clinical practice to treat patients with flow-limiting atherosclerosis. However, stenting is limited by restenosis, a homeostatic response to vascular injury, which leads to the recurrence of symptoms. Balloon inflation at high pressures and the foreign metallic stent struts lead to loss of the endothelium with subsequent smooth muscle cell (SMC) proliferation and matrix deposition leading to luminal narrowing after the procedure. It has been shown that accelerated re-endothelialization by mature endothelial cells (ECs) effectively inhibits SMC migration, proliferation, and neointima formation, and therefore, prevents the development of the early stages of restenosis after vascular injury.1 In recent years, increasing evidence indicates a repairing capacity of endothelial progenitor cells (EPCs),2,3 providing a novel cell therapeutic option for various vascular diseases.
Embryonic stem cells (ESCs) are a promising source of pluripotent stem cells. They have the capacity for unlimited growth and self-renewal and the ability to differentiate into all types of mature tissue cells. In the past several years, accumulating evidence indicates that ES cells can differentiate into ECs in vitro or in vivo.4–8 Methods to produce large number of ECs with high purity from ES cells in vitro are lacking, and little is known about the mechanism of EC differentiation and the therapeutic potential of ESC-derived ECs in cardiovascular diseases.
ESCs undergo complex gene-specific and functionally important remodeling of chromatin structure necessary for differentiation. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are critical to these processes regulating the structure and function of chromatin. Inhibition of HDACs is reported to block tumor angiogenesis,9–11 and prevent ESC differentiation,12 indicating that some HDACs are involved in ESC differentiation. Modulation of HDACs to promote production of endothelial cells from ESCs is unexplored.
Our previous data showed that stem cell antigen-1 (Sca-1) can be used as a sorting marker for vascular progenitors to allow isolation of these cells from adult adventitial tissues.13 We hypothesized that Sca-1+ progenitor cells derived from ESCs can serve as EC progenitors leading to ECs in vitro, and furthermore that HDAC3 is important in EC differentiation from Sca-1+ progenitor cells. Because endothelial dysfunction plays an essential role in vascular diseases, we also postulate that local transplantation of ESC-derived ECs (esECs) have therapeutic potential as a strategy to enhance the biological function of reconstituted endothelium. In the present study, we isolated Sca-1+ cells from mouse ES cells, differentiated these progenitors into ECs, and characterized these cells in detail. We have successfully prepared large numbers of ECs with high purity from ES cells, and evaluated the therapeutic effect of ECs on re-endothelialization and neointima formation in a mouse model of wire induced femoral artery injury.
Materials and Methods
The detailed materials and methodology for vascular endothelial growth factor (VEGF) treatment, trichostatin A (TSA) treatment, histone deacetylase activity assay, histology, chromosome detection, and X-gal staining for detection of LacZ, Western blot analysis, Uptake of DiI-Ac-LDL, double-immunofluorescence staining, flow cytometry analysis, esEC-induced angiogenesis in vivo, morphometric analysis, and quantification of lesion formation are available online (please see http://atvb.ahajournals.org).
ES Cell Culture, Cell Sorting, and EC Differentiation
Mouse ES cells (ES-D3 cell line; ATCC, Manassas, Va) were maintained as described previously.14 ES-D3 cells were cultured on type IV mouse collagen-coated (Trevigen, Gaithersburg, Md) flasks in basic differentiation medium (DM) (α-minimal essential medium [α MEM]; Gibco), supplemented with 10% fetal calf serum (Gibco) and 0.05 mmol/L 2-mercaptoethanol (Sigma) for 3 to 4 days. As described in previous studies,13 3 to 10×105 of stem cell antigen-1-positive cells (Sca-1+ cells) were sorted from 1.0×107 of cultured ES cells by magnetic labeling cell sorting (MACS) with anti-Sca-1 immunomagnetic microbeads (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany). To obtain higher numbers of Sca-1+ cells (>5×106), these cells were expanded in ES cell culture medium for 5 to 7 population doublings. For EC differentiation, Sca-1+ cells were plated on collagen IV-coated dishes or flasks, and cultured in EC-differentiated medium [basic DM plus 10 ng/mL VEGF165 (Bender MedSystems, Vienna, Austria) for 3, 7, and 9 days, or passaged every 3 days to 21 days for longer EC differentiation.
Plasmids and Transient Transfection
Reporter systems for CD31, CD144, endothelial nitric oxide synthase (eNOS), Flk-1, Flt -1, and von Willebrand factor (vWF) were cloned into pGL3-Luc vectors, and confirmed by DNA sequencing. For transient transfection, Sca-1+ cells were cultured in collagen IV-coated 12-well plate for 4 days, then transfected with reporter gene (0.4 μg/well) alone or together with HDAC3-siRNA (0.067 nmol per well). pRL-TK vector (Promega; 50 ng per well) was included in all transfection assay as internal control, and control siRNA #4611 was used as mock control; 48 hours later, the transfected cells were subjected to 20 ng/mL VEGF treatment for 12 hours or 24 hours. Dual luciferase activity assay was performed at 72 hours after transfection, and detected with a commercial kit (Promega). Relative luciferase unit was defined as the ratio of luciferase activity of specific gene to Renilla luciferase activity with that of control set as 1.0.
Adenoviral HDAC3 Gene Transfer
HDAC3 cDNA was amplified from embryonic stem cell with a primer set of 5′-atgacc ggtaccgtggcgtatttctacgac-3 and 5′-cacagcaagcttgctgctctaaatctccac-3′, and cloned into KpnI/HindIII sites of pShuttle2-Flag vector, creating the plasmid pShuttle2-Flag-HDAC3, which was verified by sequencing. Ad-Flag-HDAC3 was created from pShuttle2-Flag-HDAC3 with Adeno-XTM expression system (PT3414–1; Clontech, Mountain View, Calif) and virus was produced, amplified, and titered according to protocol provided. For adenoviral gene transfer, the Sca-1+ cells were cultured in DM for 4 days and infected with Ad-HDAC3 or Ad-TTA with 5 or 10 multiple of infection (MOI) for additional 3 days, followed by Western blot analysis. The Ad-TTA virus was used as control and to compensate the MOI.
siRNA Knockdown Experiment
The control siRNA (#4611) and the siRNA for HDAC3 (5′-ccucaucgccuggcauugatt-3′ and 5′-ucaaugccaggcgaugaggtt-3′) were purchased or synthesized from Ambion Ltd (Huntingdon, Cambridgeshire, UK). For siRNA knockdown experiments, Sca-1+ cells were cultured on collagen IV-coated plates for 3 days, and the medium was refreshed at 24 hours and 1 hour before transfection; 10 μL of 10 mmol/L siRNA per well (0.2 nmol per 10 cm2) was introduced into the cells with siIMPORTER transfection reagents (Upstate) according to the protocol provided. The transfected cells were further cultured for 48 hours, subjected to VEGF treatment for 24 hours.
Mouse Femoral Artery Denudation Injury and Cell Delivery
ApoE-deficient mice were anesthetized and the surgical procedure was similar to that described previously.15,16 Removal of the endothelium of the femoral arteries was achieved by 3 passages of a 0.25 mm angioplasty guide wire (Brivant Ltd, Ireland). After removal of the wire, the femoral artery was temporarily clamped at the level of the inguinal ligament, then 50 μL medium with or without esECs with passage number 5 to 10 (5×105), which means that esEC were cultured for 21 days or more in the presence of VEGF, was injected into the injured vessel and ligated. After 30 minutes of incubation, the clamp was removed, and the wound was closed. In some experiments, esECs were prelabeled with adenovirus-LacZ virus (MOI: 20) to trace these cells in vivo.
Data expressed as the mean±SEM were analyzed with a 2-tailed Student t test for 2 groups or pair-wise comparisons. A value of P<0.05 was considered to be significant.
Characterization of ESC-Derived ECs
The number and purity of differentiated ECs determine their potential as cell-based therapy in clinical medicine. To produce large numbers of ECs with high purity from ESCs, Sca-1+ cells were treated with 10 ng/mL VEGF in DM for 21 days or more, subcultured with a ratio of 1 to 3 every 2 to 3 days (esECs) (supplemental Figure IA, available online at http://atvb.ahajournals.org). Morphologically, ESCs displayed clusters in an undifferentiated status for >35 passages in our culture conditions, whereas Sca-1+ progenitors and esECs displayed a monolayer in culture. In addition, Sca-1+ cells grew as round cells, and esECs displayed typical “cobblestone” morphology (supplemental Figure IB). esECs, like ESCs, had a normal content of chromosomes, ie, 39.8±1.5, indicating cell fusion is not involved during cell differentiation (supplemental Figure IC). This technique provides the ability to produce large numbers of cells (≈3 to 6 ×108,9) because the population doubling time of esECs was 2.5±0.50 days and the number of population doublings of esEC reached 35±5.5 60 days after differentiation.
Previously we have shown that up to 35 passages ES cells are SSEA-1–positive but Sca-1–negative, whereas almost all Sca-1–positive cells are SSEA-1–negative.17 When isolated Sca-1+ cells were treated with 10 ng/mL of VEGF165 for 21 days or more, the majority of esECs were positive for EC linage-specific markers as demonstrated by immunofluorescence staining, with Flt-1 (Figure 1A; Flt-1), Flk-1 (Figure 1A; Flk-1), vWF (Figure 1A; vWF), CD31 (Figure 1A; CD31), CD106 (Figure 1A; CD106), and CD144 (Figure 1A; CD144). To validate the specificity of these antibodies, adult mature human umbilical vein endothelial cells (HUVECs) (positive control), adult mature aortic SMCs (negative control), and IgG control for individual primary antibodies were stained. Importantly CD31, vWF, and CD144 were expressed on HUVECs in the typical pattern, but not aortic SMCs (supplemental Figure IIA). FACS analysis demonstrated that 97.5% of ESCs were SSEA-1–positive and 32% CD31-positive (supplemental Figure IIB), yet Sca-1 and VE-cadherin–negative. After 3 or 4 days of culture in collagen-coated flasks, a small fraction of cells (8.6%) were Sca-1+, whereas the majority of cells (94.7%) isolated with magnetic beads coupled with anti–Sca-1 antibodies were Sca-1+.17 Expanded Sca-1+ cells (passage 5 to 10) expressed some progenitor cell markers, eg, Flk-1 (32.6%), but not mature EC markers (CD106 and CD144). When Sca-1+ cells were treated with 10 ng/mL of VEGF165 for 7 days, some mature EC linage-specific markers were expressed (Figure 1B; esEC-d7). As expected, the percentage of cells expressing mature EC markers further increased after 21 days or more of VEGF treatment, including CD31 (from 74.5% to 98.7%), CD106 (from 16.8% to 46.7%), CD144 (from 5.8% to 57.3%), Flt-1 (from 44.5% to 64.5%), and vWF (from 21.5% to 74.8%) (P<0.05), whereas the percentage of progenitor markers decreased, ie, Flk-1 (from 39.8 to 16.8%) (table in Figure 1B; supplemental Figure IIC). Lineage markers for nonendothelial cells, such as SMCs (smooth muscle a-actin [SMA]), T lymphocytes (CD3), common white blood cell antigen (CD45), and macrophages (Mac-1), did not or were very weakly expressed in esEC-d21 as demonstrated by FACS analysis (supplemental Figure IIC). To further characterize these esECs, they were incubated with DiI-acLDL. esECs rapidly took up DiI-acLDL and expressed high levels of CD31 (Figure 1C; supplemental Figure IID), whereas mouse SMCs did not uptake DiI-acLDL and were negative for CD31 (data not shown). To address the difference between esECs and mature adult ECs, which do not significantly contribute to neoangiogenesis and re-endothelialization in adult organisms, clonogenic potentials of Sca-1+ cells, esEC-p8, and HUVEC-p8 were assessed. Cells (2500) were re-plated into 6 well-plates coated with collagen IV and cultured for 6 days, followed by fixation and staining with hematoxylin and eosin. As shown in Figure 1D, the clonogenic potential of esECs is much higher than that of HUVECs, but lower than Sca-1+ cells (P<0.05).
Differentiaton of ECs From VEGF-Induced Sca-1+ Cells Is Dependent on HDAC
VEGF promoted Sca-1+ cells to differentiate into ECs, demonstrated by transfection assays using reporter genes that showed VEGF promoted EC lineage-specific gene expression of CD31, CD144, eNOS, vWF, and Flt-1 (Figure 2A) In keeping with these findings, protein levels of EC-linage-specific markers including CD31, eNOS, Flt-1, and Flk-1 were increased (Figure 2B), whereas Sca-1 protein levels decreased in a time-dependent manner. In the same experiment HDAC3 protein levels were increased in response to VEGF stimulation.
Published data showed that prevention of global histone deacetylation by treatment with TSA prevents ES cell differentiation.12 To explore the role of HDACs in VEGF-induced EC differentiation, we used TSA in further VEGF treatment experiments. 50 nM TSA significantly abolished VEGF-induced HDAC activation (supplemental Figure IIIA). Furthermore, the increased protein levels of the EC-lineage markers CD31, Flk-1, and Flt-1 induced by VEGF were abolished by TSA (Figure 3A). To confirm the role of HDAC3 in EC differentiation, we overexpressed HDAC3 in Sca-1+ cells using adenoviral gene transfer, followed by Western blot analysis. Overexpression of HDAC3 increased protein levels of Flk-1, Flt-1, and eNOS protein level in a dose-dependent manner (Figure 3B). These results suggest that upregulation of HDAC3 could promote Sca-1+ cells differentiating into ECs.
To determine whether HDAC3 is essential for VEGF-induced EC differentiation, we performed siRNA-mediated knockdown experiments. Knockdown of HDAC3 reduced protein levels of Flt-1, Flk-1, and eNOS (Figure 3C). In addition, luciferase reporter assays revealed that HDAC3 siRNA treatment inhibited CD144, eNOS, vWF, Flk-1, and Flt-1 gene expression under control conditions, and also reduced VEGF-induced CD31, CD144, eNOS, vWF, and Flt-1 gene expression (Figure 3D; supplemental Figure IIIB). These results indicate that HDAC3 is essential for VEGF-induced EC differentiation from Sca-1+ progenitor cells.
Angiogenesis In Vivo
To investigate whether esECs have angiogenic functions in vivo, esECs were mixed with Matrigel, injected into C57BL/6J mice and harvested at 1, 2, 3, or 4 weeks after the procedure. As shown in Figure 4A to 4F, esECs formed various highly vascularized structures including capillary-like networks (Figure 4A), micro (<20 μm; Figure 4B), small (25 to 50 μm; Figure 4F), medium (50 to 100 μm; Figure 4C) and large (>100 μm; Figure 4D) vessels. To determine the origin of cells constituting the neovascularization within the implant, we prelabeled esECs with Adenovirus expressing LacZ before implantation. Most neovessels were LacZ-positive, indicating an esEC origin of these neovessels (Figure 4E and 4F). Quantitative analysis of X-gal staining for LacZ expression showed that (81.5±12.6%) of neovessels originated from esECs. Double staining for β-gal and CD144 demonstrated that most cells lining the vessels were double-positive (Figure 4G to 4I). The EC-specific markers were expressed largely on the luminal surface of neovessels. Interestingly, double positive staining for β-gal and SMC-specific markers, such as smooth muscle myosin heavy chain, were not observed in the present study (data not shown). These findings indicate that esECs do not contribute to the formation of perivascular smooth muscle, and that perivascular SMCs that participate in the formation of neovessels may be recruited from the host. Importantly, no tumor-like tissues formed from cell implantation at any time point (1 to 4 weeks). These results strongly suggest that these esECs have high purity and angiogenic function in vivo.
Local Transfer of esECs Inhibits Neointima Formation
To evaluate the in vivo effect of esECs on neointima formation after arterial injury, mice were given sham-treatment (medium alone) or local esEC transfer after femoral artery wire induced injury. Figure 5 shows representative photographs of injured vessels, and quantitative morphometric measurements. Wire-induced arterial injury resulted in prominent neointima formation (Figure 5B), which was markedly inhibited by local injection of esECs (Figure 5C and 5D). Morphometric analysis revealed that local transfer of esECs significantly reduced neointima formation (8036 μm2±866 μm2 versus 30 026 μm2±2500 μm2, P<0.001) as well as the ratio of neointima/total vessel wall (0.198±0.06 versus 0.9±0.08, P<0.01) (Figure 5E).
When adenovirus-LacZ–infected esECs were applied to injured arteries, β-gal–positive cells were detected on the majority of the luminal surface (Figure 6B), and in some areas showed confluence (Figure 6C). No β-gal–positive cells were observed in mice given medium alone (Figure 6A), indicating that transferred esECs were incorporated into the injured arterial site. Immunofluorescence staining confirmed double-positive cells for β-gal and EC-specific markers, such as CD144 (Figure 6D to 6F) and vWF (Figure 6G to 6I). Luminal immunofluorescence intensity showed that local esEC transfer significantly increased re-endothelialization of injured arteries up to 86%±13.6%. These results further validate the finding that locally applied esECs contribute to re-endothelialization of injured arteries.
In the present study, we report a technique to produce large numbers of endothelial cells from embryonic stem cells (≈3 to 6×108,9 ECs from 107 ES cells), which may be used as therapy after vascular injury. We observed, first, that Sca-1+ progenitor cells derived from ES cells can differentiate into ECs with high purity, and the mechanism for this involves induction of HDAC3. Second, after incorporation into Matrigel esECs form vascular-like structures in vivo. Finally, local application of esECs to areas of vascular injury promotes re-endothelialization and retards neointima formation. These findings strongly suggest that Sca-1+ progenitor cells derived from embryonic stem cells may be used to form endothelial cells. These esECs have therapeutic implications for vascular diseases, and may be a promising cell source for cell-based vascular engineering and repair of injured vessels.
Several studies have reported that ES cells can spontaneously undergo vascular differentiation through first forming embryoid bodies in vitro.14 These ES cells were differentiated into ECs by growth factor cocktails, including VEGF, basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor.7 Another group reported that Flk-1+ cells derived from ES cells can differentiate into smooth muscle cells and ECs in vitro.4–6 In most studies, ES cell-derived ECs are cell mixtures limiting their use for studying the functional role of stem cell-derived ECs. In the present study, we sorted Sca-1 progenitor cells from ES cell cultures, and demonstrate that Sca-1 may be used as a sorting marker for the isolation of vascular progenitor cells. These findings suggest that this novel technique is useful for obtaining large numbers of ECs with high purity.
A panel of genetic, antigenic and functional assays is required to provide optimal characterization of EC populations differentiated from primary ES cells. We demonstrated that esECs have EC functions based on the following observations. First, esECs display typical “cobblestone” endothelial cell morphology in culture. Second, esECs express high levels of mRNA of almost all the EC markers, including CD31, CD62E, CD105, CD106, CD109, CD141, CD144, CD146, Flk-1, Flt-1, Tie-1, Tie-2, and vWF (data not shown). Furthermore, immunofluorescence and FACS analysis show that esECs are positive for EC lineage-specific markers such as CD31, CD106, CD144, Flk-1, Flt-1, and vWF. We found that Flk-1 expression decreased with EC differentiation (Figure 1B) that may be attributed to higher expression of Flk-1 in progenitors.4–6 Interestingly, high levels of CD31 mRNA or protein were unexpectedly detected on undifferentiated ES cells by reverse-transcription polymerase chain reaction, immunofluorescence staining, flow cytometry analysis, and Western blot analysis. Although CD31 is accepted as one of the main markers of ECs and platelets, this observation may reflect a possible expression of CD31 in the inner cell mass of blastocytes. Importantly, our results are consistent with some previous findings. Vittet et al found mouse ES cells are Tie-2 and CD31-positive regardless of culture conditions.14 Third, almost all esECs are positive for CD31 and rapidly uptake Dil-acLDL, indicating CD31-positive esECs in our experiment show some EC characteristics. Fourth, in comparison to mature adult ECs, esECs have much higher clonogenic potential (Figure 1D), possibly providing improved ability to influence neoangiogenesis and re-endothelialization in the adult organism. Finally, vascular-like structures including capillary networks and vessels of various sizes were observed in the in vivo angiogenesis experiments. It is thus likely that ECs derived from ESCs share a similar phenotype and ability to function as ECs generated from adult progenitors and the vascular wall.
Previous studies have specified a number of molecules involved in vascular development. Among these molecules, VEGF,18 fetal liver kinase-1 (also known VEGF-R2),19 fms-like tyrosine kinase (Flt-1, also known VEGF-R1),20 and VE-cadherin (also known CD144)21 have been reported as essential for vasculogenesis. In particular, VEGF plays a critical role in endothelial proliferation, differentiation, and vascular development.22,23 However, little is known about the mechanism by which VEGF promotes Sca-1+ cells to differentiate into ECs. Recently, Ajamian et al reported that mRNA of class II histone deacetylases increased during neural differentiation.24 Inhibition of HDAC activity by Trichostatin A blocked tumor angiogenesis9–11 and prevented ES cell differentiation.12 In another study by Rossig et al, shear stress activated class I HDACs, which was essential in shear-induced EC differentiation in human EPCs.25 In our study, we showed that VEGF increased HDAC3 protein levels and that inhibition using TSA and siRNA knockdown or upregulation with adenovirus modulated EC differentiation. These findings demonstrate for the first time to our knowledge that HDAC3 is critical for VEGF-induced EC differentiation from Sca-1+ cells.
The ultimate goal of understanding stem cell biology is the potential for using these cells therapeutically in clinical medicine. Accumulating evidence indicates that adult progenitor cells, such as bone marrow cells26–28 and endothelial progenitor cells,29–31 have beneficial effects on neovascularization following ischemia and re-endothelialization after vascular injury thereby providing a potentially novel therapeutic option for vascular disease states. In the present study, we found lesions in ApoE−/− mice were more severe and appeared earlier than in wild-type mice. This finding may explain why almost all injured arteries without cell treatment were occluded by neointimal lesions 2 weeks after injury. esEC transfer significantly enhanced re-endothelialization and reduced neointima formation in denuded vessels. Therefore, esEC-based therapeutic strategies may be a treatment option for prevention of vascular diseases such as atherosclerosis, restenosis, and graft failure.
Previous studies have shown that systemic transfusion of adult progenitor cells have beneficial effects on neovascularization after ischemia and re-endothelialization after endothelial injury.26–31 We first used this strategy in our study but found esECs provided no significant benefit on re-endothelialization compared with sham treatment. Compared with systemic application, we found a striking beneficial effect of esECs on re-endothelialization after local application (supplemental Figure IV). These findings strongly suggest the use of different approaches in experimental and clinical cell therapy trials.
In conclusion, we have established a novel method for producing large numbers of ECs from ES cells, which regenerated the endothelium of denuded vessels and reduced neointimal lesion formation after arterial injury. HDAC3 was critical in VEGF-induced EC differentiation from Sca-1+ cells. These findings provide important details on targeting proteins that promote endothelial differentiation and activation, and provide direct evidence to support stem cell-based therapeutic approaches.
We thank Dr Neil Roberts and Evelyn Torsney for critical reading of the manuscript.
Sources of Funding
This work was supported by grants from the Oak Foundation and British Heart Foundation.
Original received April 6, 2006; final version accepted July 25, 2006.
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