Published online before print August 14, 2008, doi: 10.1161/CIRCRESAHA.107.166405
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© 2008 American Heart Association, Inc.
Molecular Medicine |
E-Cadherin/β-Catenin/T-Cell Factor Pathway Is Involved in Smooth Muscle Cell Proliferation Elicited by Oxidized Low-Density Lipoprotein
From the Institut National de la Santé et de la Recherche Médicale U858 (A.B., A.N.-S., M.-H.G., J.-C.T., R.S., F.M.-S.), Toulouse, France; Faculty of Medicine-Rangueil (A.B., A.N.-S., M.-H.G., J.-C.T., R.S., F.M.-S.), University Paul-Sabatier Toulouse III, Toulouse, France; and Department of Pathology (S.H.), Cardiovascular Research Institute Maastricht, University of Maastricht, The Netherlands.
Correspondence to Dr F. Maupas-Schwalm. Biochimie, INSERM U858, IFR-31, CHU Rangueil 1, Avenue Jean Poulhès, TSA-50032-31059 Toulouse Cedex 9, France. E-mail schwalmf{at}toulouse.inserm.fr
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Abstract |
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The E-cadherin/β-catenin/T-cell factor (Tcf) signaling pathway plays a crucial role in embryogenesis and carcinogenesis and has recently emerged in atherosclerosis. The aim of this work was to investigate whether this signaling pathway is involved in smooth muscle cell proliferation induced by oxidized low-density lipoprotein (LDL). In human aortic smooth muscle cells, mitogenic concentration of mildly oxidized LDL induced the activation of β-catenin, as assessed by the dissociation of the β-catenin/cadherin complex, and the concomitant rise of active β-catenin in the cytosol. The oxidized LDL–induced rise of active β-catenin required metalloproteinase activation, as well as epidermal growth factor receptor and Src signaling, as assessed by the use of pharmacological inhibitors and cells overexpressing a SrcK-inactive form. The concomitant phosphatidylinositol 3-kinase/Akt activation and glycogen synthase kinase 3-β phosphorylation induced the inhibition of the proteasomal degradation of β-catenin. Then active β-catenin associated with Tcf4 and translocated into the nucleus. This enhanced the expression of the cell cycle activator cyclin D1. This crucial role of β-catenin in the mitogenic effect of oxidized LDL was confirmed by silencing β-catenin by specific small interfering RNA that blocked DNA synthesis. Immunohistochemistry staining of stable and disrupted plaques from carotid endarterectomy sections showed a correlation between active β-catenin and Ki67, a proliferation marker, and a more intense staining in the smooth muscle cell layer surrounding the lipid core of disrupted plaques. In conclusion, the β-catenin pathway is required for the mitogenic effect of oxidized LDL on human aortic smooth muscle cells. This study highlights the putative important role of the E-cadherin/β-catenin/Tcf signaling pathway in atherosclerosis.
Key Words: atherosclerosis • oxidized LDL • β-catenin signaling pathway
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Atherosclerosis arises from a multifaceted pathophysiology characterized by chronic inflammation, ubiquitous accumulation of lipids, and vascular cells modifications in the arterial wall.1 Development of lesions starts with endothelial dysfunction, leading to plaque formation and finally to plaque disruption, which exposes the lipid core to blood and initiates thrombus formation and artery flow disruption in a vicious circle between inflammation and thrombosis.2 The concept of unstable atherosclerotic plaques asks a crucial question.2–3 Plaque instability is dependent on fibrous cap thickness, which is itself dependent on the smooth muscle cells (SMCs) migration and proliferation.4 SMC proliferation contributes to the extension of atherosclerotic plaques but, on the other hand, could strengthen the fibrous cap and protect the artery vasculature of instability and plaque rupture. Many biological agents regulate SMC proliferation.1,4–6 It is admitted that low-density lipoprotein (LDLs), with their susceptibility to oxidative damage, exert a significant role in atherogenesis.5,7 Oxidized LDLs (oxLDLs) have a biphasic effect on SMCs, ie, mitogenic at low concentration, apoptotic at high concentration, and are able to trigger several signaling cellular pathways, mediated by tyrosine kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, metalloproteinases, and sphingolipids.8–10 Nevertheless, signaling pathways activated by mitogenic oxLDL are only partly known.
The catenin pathway, particularly conserved during the evolution, is a crucial actor of embryogenesis, cancer diseases, and actually emerged in several inflammatory diseases.11 Catenins (
-catenin, β-catenin, plakoglobin, and p120 catenin) belong to the Armadillo family proteins. They are members of cell-adherent junctions and bind the cytosolic tail of cadherins.12 They contribute to stabilize cell adhesion and stow the cytoskeleton to the membrane.13,14 β-Catenin and plakoglobin act as a bridge connecting the E-cadherin to -catenin, which links actin filaments.15 The cadherin–catenin complex is a target for many cell signaling pathways (for instance tyrosine phosphorylation by growth factors receptors16) involved in adhesion, proliferation, and cell motility.17 β-Catenin acts as mediator of the Wnt pathway involved in cell proliferation and differentiation during embryogenesis or development of cancers.11 Usually, the β-catenin turnover is regulated in the cytosol by a large protein complex including adenomatous polyposis coli (APC)/axin/creatine kinase-1/glycogen synthase kinase (GSK)3-β in which GSK3-β promotes the β-catenin ubiquitination and its subsequent proteasomal degradation. When β-catenin is released in the cytosol and is not degraded by the proteasome, it may translocate to the nucleus as a complex with T-cell factor (Tcf). The β-catenin/Tcf complex acts as a transcriptional activator of many genes,18 such as cyclin D1 gene.
Recent studies report the activation of the β-catenin pathway by growth factors in vascular smooth muscle cells.19,20 Nevertheless, the role of the E-cadherin/β-catenin/Tcf pathway in atherogenesis is only poorly understood.
Our study attempts to clarify the putative role of the β-catenin pathway in the mitogenic effect of oxLDL on human aortic SMCs. This should consequently highlight a crucial role of the β-catenin pathway in the development of atherosclerotic lesions.
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Materials and Methods |
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Human LDL (1.019_d_1.063) were isolated by sequential ultracentrifugation and oxidized by UV-C irradiation. We used human aortic smooth muscle cells (CRL 1999, American Type Culture Collection) and SrcK+ and SrcK– cells, derived from C3H-10T1/2 murine embryo-transfected fibroblasts. The following methods were used: subcellular fractionation, immunocytochemistry, immunoprecipitation, Western blot analysis, [H3]thymidine incorporation, fluorometric determination of MT1–matrix metalloproteinase (MMP) activity, and knockdown of protein by small interfering (si)RNA transfection. Human tissues were obtained by carotid endarterectomy and used for immunohistochemistry or Western blot analysis after crushing. The SigmaStat software was used for statistical analysis.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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Activation of β-Catenin by oxLDL in Human SMCs
Human LDL were oxidized by UV (oxLDL, 9.7±1.5 nmol of malondialdehyde [MDA] per milligram of apolipoprotein [apo]B; native LDL, 0.5±0.2 nmol MDA per milligram of apoB) (Figure 1A). Under the used conditions, a moderate concentration of oxLDL (50 µg apoB/mg) was mitogenic, whereas native LDL had no effect on SMC proliferation, as shown by [3H]thymidine incorporation and MTT assay (Figure 1B), in agreement with previous studies.21 OxLDL induced β-catenin activation (peaking at 60 to 90 minutes), whereas native LDL (LDL) did not (Figure 1C). Pretreatment of cells by LiCl (20 mmol/L, 6 hours) was used as a positive control. The absence of β-catenin activation by native LDL supported the specific effect of oxLDL.
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Modification of β-Catenin Subcellular Localization Induced by oxLDL in Human SMCs
After stimulation with oxLDL, β-catenin dissociated from E-cadherin (shown in the Western blot of β-catenin immunoprecipitate [IP], determined by using anti–E-cadherin) (Figure 2A) and associated with Tcf4 (shown by Tcf4 IP, determined by anti–β-catenin) (Figure 2B). The time course of subcellular localization of β-catenin showed a progressive decrease in the cytosolic fraction and a concomitant significant increase in the nuclear fraction (Figure 2C and 2D), thus suggesting that active β-catenin (as Tcf4/active β-catenin complex) translocated into cell nucleus. These results were also supported by confocal microscopy (Figure 2E). In untreated cells (Figure 2E, left images), β-catenin was mainly localized at the cell membrane, with a low level of active form in the cytoplasm and in the nucleus. After 1 hour of treatment with oxLDL (Figure 2E, right images), the distribution of β-catenin in subcellular compartments was altered, as shown by the nuclear translocation of the active form. This reveals that oxLDL are able to induce β-catenin activation and subsequent translocation of the active form, as a complex with Tcf4, into the nucleus.
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MT1-MMP Activation by oxLDL in Human SMCs
OxLDL are able to activate MMPs in vascular cells.9 According to these previous results, mitogenic concentration of oxLDL (50 µg apoB/mL) induced the activation of the membrane-bound metalloproteinase MT1-MMP peaking at 30 minutes (Figure 3A). As expected, MT1-MMP activation by oxLDL was inhibited by Marimastat, a broad-spectrum MMP inhibitor (Figure 3B) and by a MT1-MMP–specific siRNA (under the used conditions, MT1-MMP was downregulated by 70%) (Figure 3C and 3D).
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MT1-MMP Is Required for oxLDL-Induced β-catenin Activation
Because MMPs participate in extracellular matrix turnover and E-cadherin degradation,22 we investigated the implication of MT1-MMP activation in the oxLDL-induced β-catenin activation. OxLDL induced a progressive decrease of cellular E-cadherin (Figure 4A and the online data supplement [Figure IA]), which was inhibited by Marimastat (Figure 4B and supplemental Figure IB) and MT1-MMP–specific siRNA (Figure 4C and supplemental Figure IC). Concomitantly, the oxLDL-induced activation of β-catenin was abrogated by both Marimastat (Figure 4B and supplemental Figure IB) and MT1-MMP siRNA (Figure 4C and supplemental Figure IC).
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These data suggest a crucial role of MT1-MMP in the decrease of E-cadherin and the activation of β-catenin by oxLDL.
β-Catenin Activation by oxLDL Is Mediated by EGFR and c-Src Tyrosine Kinases
The mitogenic effect of oxLDL requires the activation of the EGF receptor (EGFR)8 and of Src family kinases.23 Because β-catenin is a potential target of these kinases,24 we investigated whether these tyrosine kinases were required for β-catenin activation induced by oxLDL.
OxLDL (50 µg apoB/mL) induced tyrosine phosphorylation of EGFR and Src at 5 minutes, which were inhibited by AG1478 and PP2, 2 pharmacological inhibitors specific for EGFR and Src, respectively (Figure 5A). Interestingly, each tyrosine kinase inhibitor (PP2 and AG1478) inhibited the oxLDL-induced activation of both Src and EGFR, thereby suggesting cooperation between Src and EGFR, in agreement with recently reported data.25,26
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β-Catenin activation by oxLDL was also blocked by either PP2 or AG1478 (Figure 5B and 5C), thus suggesting a role for the EGFR/Src pathway in β-catenin activation. To confirm the involvement of Src in β-catenin activation, we used a construct expressing a dominant-negative kinase defective form of Src (SrcK–). In SrcK- fibroblasts, oxLDL did not trigger activation of β-catenin, in contrast to SrcK+ cells (wild-type fibroblast expressing the active form of Src) (Figure 5D).
Taken together, these data suggest that β-catenin activation by oxLDL is dependent on EGFR/Src signaling.
Decrease of β-Catenin Proteasomal Degradation by oxLDL
When β-catenin dissociates from the cytosolic tail of E-cadherin, the free β-catenin released in the cytosol may either be degraded by the proteasome or translocate into nucleus. This is dependent on APC/axin/GSK3-β complex, in which the constitutively active GSK3-β phosphorylates β-catenin and promotes its degradation by βTrCP targeted ubiquitin/proteasome pathway.11 Degradation of β-catenin is suppressed by the phosphorylation/inhibition of GSK3-β by Akt/PKB, which is activated by growth factor receptors (eg, EGFR)/PDK/PI3K pathway.
OxLDL induced the phosphorylation of Akt and of GSK3-β (peaking at 30 minutes) (Figure 6A). The PI3K specific inhibitor LY290042 inhibited Akt and GSK3-β phosphorylation by oxLDL, thus confirming that GSK3-β phosphorylation/inhibition requires the activation of PI3K/Akt pathway by oxLDL (Figure 6B). As expected, LY290042 also reduced β-catenin activation triggered by oxLDL (Figure 6C and 6D). Moreover, the proteasome-specific inhibitor PSI abolished the loss of activated β-catenin induced by LY290042 (Figure 6C and 6D).
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Taken together, these data reflect the capacity of mitogenic oxLDL to inhibit the proteasomal degradation of β-catenin via GSK3β phosphorylation/inhibition mediated by the PI3K/Akt pathway.
Involvement of β-Catenin Activation in the oxLDL Mitogenic Effect on SMCs
On bypassing the destruction machinery, β-catenin binds to Tcfs, translocates to the nucleus, and recruits the chromatin-remodeling proteins p300 and Brg-1 to responsive promoters, thereby activating the transcription of specific target genes, including a cyclin D1, and inducing cell cycle progression.19 This led us to investigate whether β-catenin was required for upregulation of cyclin D1, proliferating-cell nuclear antigen (PCNA), and Ki67 (nuclear proteins that are expressed in proliferating cells) by oxLDL.
OxLDL (50 µg apoB/mL) induced a significant increase of cyclin D1 between 2 and 8 hours (Figure 7A and supplemental Figure IIA). Silencing β-catenin by a specific siRNA (siRNA β-cat), which abolished both the total and the active form of β-catenin (supplemental Figure IIB), inhibited the oxLDL-induced rise of cyclin D1 (Figure 7B and supplemental Figure IID). In the same way, the knockdown of the β-catenin coactivator Tcf4 by a specific siRNA (50% of protein synthesis; supplemental Figure IIC) decreased significantly the rise of cyclin D1 (Figure 7B and supplemental Figure IID). In contrast, as expected, an irrelevant scrambled siRNA (sc-siRNA) had no effect on β-catenin level (data not shown) and on cyclin D1 level (Figure 7B and supplemental Figure IID). Inhibitors of the cell signaling pathways implicated in β-catenin activation (tyrosine kinases inhibitors, PP2, AG1478, PI3K inhibitor LY294002, and MT1-specific siRNA) inhibited the effect of oxLDL on cyclin D1 expression (Figure 7B and supplemental Figure IID). On confocal microscopy, immunocytochemical staining showed an increased level of nuclear expression of both PCNA and Ki67 in cells treated with mitogenic oxLDL (Figure 7C and supplemental Figure IIE). Pretreatment of cells with a β-catenin–specific siRNA (siRNA β-cat), but not with the irrelevant siRNA (sc-siRNA), abolished the oxLDL-induced nuclear expression of both proliferation markers (Figure 7C, bottom images, and supplemental Figure IIE). In the same way, siRNA specific to β-cat and Tcf4, as well as the signaling inhibitors PP2, AG1478, LY294002, and MT1-specific siRNA, inhibited [3H]thymidine incorporation induced by oxLDL (Figure 7D).
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Taken together, these results suggest a crucial role of β-catenin activation in the mitogenic effect of oxLDL.
β-Catenin Is Expressed in Human Atherosclerotic Tissues and Its Level Is Correlated With the Expression of Cellular Mitotic Markers and the Plaque Evolution
Few studies27,28 have reported a β-catenin expression in human atherosclerotic tissues. We analyzed the expression of β-catenin and its active form on Western blot of protein extracts from 5 human carotid endarterectomy segments. This revealed a noticeable expression of active β-catenin in disrupted plaques (supplemental Figure IIIA and IIIB) associated with increased expression of the mitotic marker PCNA (supplemental Figure IIIC). Immunohistochemistry staining with anti–active β-catenin antibody revealed a positive staining of SMCs in moderate intimal hyperplasia (Figure 8A, left image) and a more intense positive staining in SMC layers located under the lipid core in disrupted plaque (Figure 8A, right image). The proliferation marker Ki67 was highly expressed in disrupted plaque (sample D2) but was barely expressed in stable fibrous plaque (sample S1) (supplemental Figure IIID). In addition, the 3 immunostaining markers (active β-catenin, PCNA, Ki67) were evaluated on 14 human carotid endarterectomy sections, including 5 stable plaques (area 12.33±3.9 mm2) and 9 disrupted plaques (area 33.82±13.43 mm2) (Figure 8B and supplemental Figure IVA). The labeling of β-catenin, PCNA, and Ki67 was higher in disrupted plaques than in stable plaques (Figure 8C and supplemental Figure IVB) and, as expected, was correlated with plaque area (supplemental Figure IVC).
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When the staining of the 3 markers was evaluated per unit of plaque area, active β-catenin and Ki67 (but not PNCA) staining were significantly higher in disrupted plaques (Figure 8D).
This led us to examine whether stable plaques can be discriminated from disrupted plaques, when choosing an arbitrary threshold value for active β-catenin and for Ki67 (respectively, 3 and 1, expressed as percentage of immunostaining area to total plaque area). In our series of plaque sections, all the stable plaques were negative for Ki67 and only 20% of them were positive for active β-catenin. In contrast, 89% of disrupted plaques were positive for Ki67 and all for active β-catenin (supplemental Figure IVD). Using these arbitrary thresholds, the number of plaques positive for active β-catenin and Ki67 immunostaining was significantly different between the 2 groups (stable versus disrupted) (P=0.015), as expected. It may be noted that there is no significant difference between both immunostaining (Ki67 versus active β-catenin immunostaining, P=0.48) for the same type of plaque (supplemental Figure IVD).
Taken together, these data suggest a correlative link between the expression of active β-catenin and markers of cell proliferation in human atherosclerotic plaques, consistent with that observed on cell cultures treated with oxLDL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to previous studies,21 low concentrations of mildly oxLDL induce vascular cell proliferation, but the signaling pathways leading to the mitogenic effect are only partly known.
Our study highlights, for the first time to our knowledge, that oxLDLs induce β-catenin activation via a mechanism dependent on the activation of MMP and EGFR/Src by oxLDL (supplemental Figure V). The crucial role of β-catenin for the proliferation of human vascular SMCs induced by oxLDL is complementary and consistent with its function in vascular cell biology29–31 and supports and extends its emerging role in atherogenesis.27
Our data show that oxLDL induced the decrease of E-cadherin, the subsequent dissociation of the β-catenin/E-cadherin complex, and the release of active β-catenin in the cytosol. The activation of β-catenin is probably, at least in part, the consequence of β-catenin tyrosine phosphorylation by tyrosine kinases (such as EGFR and Src kinase) known to be activated by oxLDL.32 This β-catenin phosphorylation, already described with plasminogen activators,25 is considered as a causal mechanism of detachment of the β-catenin from the cytosolic tail of E-cadherin.17 The cadherins, beside their structural role in cell–cell homophilic interactions, are calcium-dependent cell surface receptors implicated in cell signaling leading to gene regulation.33 OxLDLs induce the release of β-catenin from its cell surface anchor protein E-cadherin, as shown by the dissociation of the E-cadherin/β-catenin complex in cells incubated with oxLDL and the decrease of E-cadherin.
Tyrosine kinases have been implicated in the regulation of intercellular adhesion involving E-cadherin, and tyrosine phosphorylation of β-catenin may regulate the cadherin-mediated adhesion.24 E-cadherin could be phosphorylated by activated Src, and this event may induce disruption of the cadherin/catenin complex.34 Despite this, Hashimoto et al have reported that oxLDL downregulated VE-cadherin on endothelial junction,35 the mechanism implicated in the oxLDL-induced β-catenin activation and in the E-cadherin loss is unknown. In cancer cells, E-cadherin deregulation may result from its internalization via integrin signaling,36 and the cell membrane turnover of E-cadherin may lead to endocytosis and degradation.37 Previous studies have reported the implication of MMPs in E-cadherin degradation,38 and E-cadherin itself has been shown to regulate MMPs expression.39,40 In our study, and according to a previous report,9 oxLDL induced metalloproteinase activation. The loss of E-cadherin and the activation of β-catenin are inhibited by Marimastat, a broad specific MMP inhibitor, and by siRNA more specific to MT1-MMP, thus suggesting that MT1-MMP is required for E-cadherin degradation and β-catenin activation induced by oxLDL. This is consistent with the role of MMPs in the degradation of extracellular matrix, thereby facilitating SMC migration and proliferation.22
PI3K should be associated with the E-cadherin adhesion complex,41 and the activation of PI3K/Akt induces the phosphorylation and the inhibition of GSK3-β that results, in turn, in reduced phosphorylation of β-catenin on serine/threonine and of its subsequent proteasomal degradation.42 We report here that the oxLDL-induced activation of PI3K/Akt survival pathway8 is required for maintaining the level of the active form of β-catenin by preventing its degradation by the proteasomal system. This effect of oxLDL was reversed by Ly294002 which inhibits PI3K and, subsequently, blocks Akt activation, GSK3 inhibition, and the subsequent increase of active β-catenin. As expected, this effect of Ly294002 was suppressed by PSI, which inhibits the proteasomal degradation of β-catenin. Moreover, it is known that Akt phosphorylates β-catenin to cause its dissociation from cell–cell contact and to promote its transcriptional activity.43
In our study, the oxLDL-activated β-catenin binds the transcriptional factor Tcf4 and translocates into cell nucleus, in agreement with a previous study.44 The mechanisms of β-catenin nuclear translocation are complex45,46 and were not analyzed here. Nevertheless, changes in β-catenin subcellular location are clues to the activation of the E-cadherin/β-catenin/Tcf pathway.47 This allows β-catenin to regulate the transcription of genes including the cyclin D1 gene.48 The inhibitory effect of β-catenin–specific siRNA on the oxLDL-induced cyclin D1 upregulation and on cell proliferation confirms the major implication of β-catenin in the mitogenic effect of oxLDL.
Finally, this work highlights a crucial role of the β-catenin pathway in SMC proliferation induced by oxLDL. The data obtained on human atherosclerotic tissues show an increased active β-catenin level in disrupted plaque associated with a more intense positive staining in SMC layers located under the core and a correlation between the expression of active β-catenin and markers of SMC proliferation. This is consistent with the hypothesis of a potential role of the β-catenin pathway in the evolution of the atherosclerotic plaque.
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Acknowledgments |
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We acknowledge Anique Janssen-Vrehen, Ine J. M. Middendorp, and Corinne Bernis for technical assistance.
Sources of Funding
This work was supported by Institut National de la Santé et de la Recherche Médicale, University Toulouse-3, grant ANR-05-PCOD-019-01 LiSA. A.B. is the recipient of a doctoral fellowship from the Institut National de la Santé et de la Recherche Médicale. S.H. participates in the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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Original received October 19, 2007; revision received July 28, 2008; accepted August 6, 2008.
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