The PPARα/p16INK4a Pathway Inhibits Vascular Smooth Muscle Cell Proliferation by Repressing Cell Cycle–Dependent Telomerase Activation
Peroxisome proliferator-activated receptor (PPAR)α, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARα activation suppresses G1→S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16INK4a (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARα is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARα activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1–dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect that was dependent on p16. The inhibition of cell proliferation by PPARα activation was lost in VSMCs following TERT overexpression or knockdown, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARα. Finally, we demonstrate that PPARα agonists suppress telomerase activation during the proliferative response following vascular injury, indicating that these findings are applicable in vivo. In concert, these results demonstrate that the antiproliferative effects of PPARα in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade.
Proliferation of vascular smooth muscle cells (VSMCs) contributes to atherosclerosis development and constitutes a primary mechanism resulting in postangioplasty restenosis, vein graft failure, and transplant vasculopathy.1 Mitogenic growth factors secreted during vascular injury converge into the cell cycle as the final common signaling pathway regulating the proliferative response of VSMCs.1 G1→S cell cycle progression is regulated by the retinoblastoma (RB)/E2F pathway, which links growth-regulatory pathways to a transcriptional program required for DNA synthesis.2 In quiescent cells, the RB family of proteins, which include pRB, p107, and p130, binds to E2F transcription factors and inhibits their transcriptional activity.2 In response to growth factors, cyclin-dependent kinase (CDK)–cyclin complexes are activated to hyperphosphorylate pRB and thereby induce the release of E2F allowing transcription of S phase genes.3 CDK inhibitors (CDKI), including p16INK4a (p16), impinge on this pathway by inhibiting the activity of cyclin–CDK complexes, providing a second layer of regulation.3
Although DNA replication is regulated through the cell cycle machinery, emerging evidence indicates that telomerase activation may affect cell proliferation by maintaining telomere function.4 Telomeres, the DNA TTAGGG repeat sequences at the ends of eukaryotic chromosomes, are stabilized by telomerase to serve as protective capping and to prevent cellular senescence.5 Telomerase activity in telomerase-deficient cells can be restored by ectopic expression of the telomerase reverse transcriptase (TERT), indicating that TERT confers the catalytic activity and is the limiting factor for telomerase activation.6 In vitro, TERT is tightly regulated by mitogenic stimuli and required for VSMC proliferation.7–9 In animal models, telomerase deficiency reduces atherosclerosis and neointima formation,10,11 indicating that telomerase may serve as a novel pharmacological target for the treatment of vascular diseases.11 However, although telomerase activity has been linked to VSMC proliferation,7–9 the mechanisms feeding into the transcriptional pathways that determine telomerase regulation in VSMCs remain to be investigated.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily.12,13 PPARα mediates the hypolipidemic effects of fibrates, which are clinically used to treat dyslipidemia in patients at cardiovascular risk.12,13 In addition to their metabolic efficacy, recent evidence has demonstrated that PPARα prevents intimal hyperplasia through direct pleiotropic effects on the vascular wall.12 Our previous work provided evidence that PPARα inhibits VSMC proliferation by inducing the expression of the CDKI p16.14 In the present study, we identify telomerase as the key downstream target for the antiproliferative effects of PPARα and demonstrate that PPARα interferes with a previously unrecognized p16/pRB/E2F-dependent transcriptional activation of telomerase in VSMCs.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Rat aortic VSMCs (rVSMCs) were cultured as described.9 Primary human coronary artery VSMCs (hVSMCs) were obtained from Lonza (Allendale, NJ) and cultured using the VSMC Growth Medium 2 Bullet Kit as directed. In all experiments, hVSMCs of passage 3 to 8 were subjected to G0/G1 phase synchronization by starvation for three days in medium supplemented with 0.4% FBS and devoid of any growth factors. Cells were pretreated for 24 hours with the indicated doses of PPARα ligand (fenofibric acid [FA], GW7647, gemfibrozil [GF], or Wy 14,643) or vehicle (Me2SO) before stimulation of cell proliferation with growth medium (GM) containing 5% FBS and growth factors (human fibroblast growth factor, human epidermal growth factor, and insulin as directed). When hVSMCs were harvested at 72 hours, the medium supplemented with PPARα agonists or DMSO was changed after 36 hours. Cell cycle distribution was determined by propidium iodide staining of DNA and flow cytometry analysis as described.14 Cell proliferation was assessed by cell counting using a hematocytometer. For all data shown, individual experiments were repeated at least 3 times in duplicates with different lots or preparations of VSMCs. For Western blot analyses, the graphical analysis represents results from three independent experiments and quantification by densitometry.
hVSMCs (1×105 cells per 6-well plates) were infected with Ad-GFP, Ad-GFP/TERT, or Ad-E2F-1 at the indicated plaque-forming units (PFU) per cell in 0.4% FBS. Transduction efficiency was above 94% in cells infected with Ad-GFP as verified by fluorescence-activated cell-sorting analysis. Infection of cells with Ad-GFP/PPARα has been described previously.14
Small Interfering RNA Analyses
Serum-deprived hVSMCs were transfected for 4 hours in Opti-MEM (Invitrogen, Carlsbad, Calif) using the indicated quantities of small interfering (si)RNAs and either 16 μL (for 60-mm2 plate) or 8.4 μL (for 6-well plates) of jetSI reagent (Polyplus Transfection, Illkirch, France). After transfection, 0.4% FBS starvation medium supplemented with the indicated PPARα ligand or vehicle (Me2SO) was added overnight. FBS and growth factors were then added to achieve the same final concentration as in the routine medium.
Telomeric Repeat Amplification Protocol Assay
Telomerase activity was analyzed using a PCR-based assay (TeloTAGGG Telomerase PCR ELISA Plus; Roche Applied Sciences) as described.9 Three micrograms of whole-cell protein were used for the elongation/amplification, and telomerase activity was quantified by ELISA according to the instructions of the manufacturer.
RT-PCR and Quantitative PCR
Transcript levels of target genes were assessed by quantitative real-time RT-PCR as previously described14 using an iCycler (Bio-Rad, Hercules, Calif), SYBR Green I system (Bio-Rad), and the specific primers indicated in the online data supplement (supplemental Table I). mRNA levels of hTBP (TATA box–binding protein) and the transcription factor mTFIIB were quantified simultaneously as house keeping gene in human and murine cells, respectively.
Western Blotting was performed as described14 following isolation of nuclear proteins using the NE-PER kit (Pierce, Rockford, Ill).
Transient Transfection Assays
rVSMCs plated in 24-well plates at a density of 5×104 cells per well were transfected for 4 to 6 hours in OptiMEM using Lipofectamine 2000 (Invitrogen) at a ratio of 2 μL of Lipofectamine 2000 per 0.8 μg of DNA. Luciferase activities were analyzed using the Dual-Luciferase Reporter Assay system (Promega, Madison, Wis). Transfection efficiency was normalized to renilla luciferase activities generated by cotransfection of 0.1 μg per well pGL4.74[hRluc/TK] (pRL-Tk, Promega).
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed using an assay kit (Millipore, Billerica, Mass) and 10 μg of antibody for the immunoprecipitation as described.14 One percent of DNA/protein extract precleared with protein G agarose was used as nonprecipitated genomic DNA (input). Extracted DNA was amplified by real-time PCR with an annealing temperature of 54°C, 200 nmol/L primer pairs that cover the proximal E2F1-binding element in the human TERT promoter (supplemental Table I), 40% pure protein/DNA-immunoprecipitated sample, and 1 mg of HotStart-IT binding protein (USB, Cleveland, Ohio). Negative controls include PCRs performed with hTERTE2F1 oligonucleotides following immunoprecipitation with nonimmune IgG or with primers covering a distal region of TERT gene localized at −899/−723 of E2F1 element (hTERTcontrol) (supplemental Table I and data not shown).
Endothelial Denudation Injury
Eight- to 12 week-old male Sv/129 mice were fed a standard chow diet (diet no 7012, Harlan Teklad, Madison, Wis) supplemented with fenofibrate (FF) or GF for 1 week before endothelial denudation injury of the femoral artery. Guide wire injuries were performed on the right femoral artery, as previously described, whereas sham surgery without injury was performed on the contralateral left side.9 Carotid artery wire injuries were performed as described.14 Mice were euthanized 2 days after injury for isolation of liver and vascular tissues. Telomerase activities were analyzed in arterial tissues by ELISA as described above. The institutional animal care and use committee at the University of Kentucky approved all procedures on the mice.
Differences between different groups were assessed by 1-way ANOVA for in vitro assays and Kruskal–Wallis for in vivo analyses of telomerase activities in femoral arteries, followed by Tukey tests. Probability values of <0.05 were considered to be statistically significant.
PPARα Agonists Inhibit Mitogen-Induced Telomerase Activity and TERT Gene Expression
To investigate the regulation of telomerase activity by PPARα ligands, mitogen-induced telomerase activity was analyzed in hVSMCs treated with different PPARα agonists, including the clinically used FA and GW7647, a PPARα agonist with high affinity and selectivity for the receptor.15 Consistent with previous reports,8,9 telomerase activity was induced following mitogenic stimulation of hVSMCs (Figure 1). Pretreatment with either FA or GW7647 resulted in a dose-dependent inhibition of mitogen-induced telomerase activation. Similar results were obtained using other PPARα ligands including GF (300 μmol/L) or Wy14,643 (100 μmol/L) (data not shown).
TERT confers the catalytic activity of telomerase,6 and we recently demonstrated that other components of telomerase, including the telomerase RNA component and the telomerase-associated protein 1, are not regulated in VSMCs.9 Because it has been suggested that TERT transcription represents the rate-limiting step for its expression,6 we next focused our analyses on the regulation of TERT gene expression by ligand-activated PPARα. As depicted in Figure 2, mitogen-induced TERT mRNA and protein expression were dose-dependently repressed by the PPARα ligands FA or GW7647 over a concentration range known to activate the receptor.15,16 Similarly, adenoviral overexpression of PPARα suppressed TERT expression, an effect that was even further pronounced in the presence of GW7647 (Figure 3). In concert, these data suggest that ligand-dependent PPARα activation inhibits telomerase activity by repressing mitogen-induced TERT transcription.
The PPARα/p16 Pathway Represses TERT Transcription and Telomerase Activity Through Inhibition of E2F-1–Dependent Trans-Activation of the TERT Promoter
To identify the regulatory elements in the TERT promoter that confer the repression by PPARα, we performed transient transfection experiments in rVSMCs using 5′-deletion series of a luciferase vector driven by the human TERT promoter region spanning from −776 to +18 bp from the transcription initiation site (Figure 4A). rVSMCs were cotransfected with a pSG5-PPARα expression vector or control plasmid and subjected to mitogenic stimulation in the presence of FA. Mitogen-induced transcriptional activity of the −776 bp TERT promoter was significantly repressed by overexpression of PPARα or treatment with FA (Figure 4A). PPARα-dependent repression of TERT promoter activity was maintained on 5′-deletion to −181 bp. In contrast, mitogenic induction and repression of TERT promoter activity by PPARα were lost on further deletion to −150 or −47 bp, indicating that a 31 bp promoter region between −181 and −150 bp confers the repression of TERT promoter activity by PPARα.
Sequence analysis of this TERT promoter region did not reveal the presence of a PPAR-response element (PPRE), suggesting an indirect mechanism responsible for the repression of the TERT promoter by PPARα. Previous reports have identified three E2F-binding sites in the proximal TERT promoter located at −170/−174, −98/−94, and +9/+14 from the transcription initiation site referred to as E2F1, E2F2, and E2F3, respectively.17,18 To further analyze their role for the regulation of the promoter, rVSMCs were transfected with a luciferase construct driven by the wild-type TERT promoter region from −267 to the immediate upstream part of the translation start codon (−267TERTLuc) or the same region bearing mutations of the E2F sites as indicated in (Figure 4B). Consistent with our observations using 5′-deletion, promoter activity of the wild-type construct was repressed by PPARα and FA. Interestingly, both the mitogen-induced TERT promoter activity and the inhibitory effect of PPARα activation were abolished on mutation of the E2F1 site (−267E2F1mTERTLuc), which is located within the −181 to −150 bp region. In contrast, the repression by ligand-activated PPARα was maintained in the construct −267E2F2mTERTLuc, although the mitogenic induction was slightly decreased (P<0.05 versus −267E2F1m). Finally, mutation of the third E2F-binding site in the TERT promoter (−267E2F3m) did not affect the transcriptional regulation.
Because PPARα agonists inhibit S phase entry by trans-activating the promoter of the gene encoding the CDKI p16,14 we next analyzed the effects of p16 on TERT promoter activity. As depicted in Figure 4C, overexpression of p16 completely suppressed mitogen-induced transcriptional activity of the wild-type −267TERTLuc construct to a level observed in quiescent rVSMCs. Similarly, as observed following PPARα activation in Figure 4B, this p16-mediated repression of the TERT promoter was dependent on a functional E2F1 site located at −170/−174. Therefore, PPARα-induced p16 expression provides a likely mechanism involved in the repression of TERT transcription by PPARα ligands.
To confirm the role of E2F-1 in the PPARα-dependent regulation of telomerase activation, we used siRNA-mediated knockdown of E2F-1 in hVSMCs (Figure 4D). E2F-1 knockdown decreased TERT mRNA expression and telomerase activity, whereas complementary results were obtained on overexpression of E2F-1 in quiescent hVSMCs (supplemental Figure I). However, in cells transfected with E2F-1 siRNA, the ability of the PPARα ligand to repress TERT transcription and inhibit telomerase activity was lost. In concert, these experiments suggest that the PPARα/p16 pathway inhibits mitogen-induced TERT expression and telomerase activity through negative cross-talk with an E2F-1-dependent trans-activation of the −170/−174 site in the TERT promoter.
PPARα Modulates Transcriptional Complexes Formed With E2F-1 and pRB Proteins at the Proximal TERT Gene Promoter
We next performed ChIP assays using primer pairs that cover the E2F element in the proximal TERT promoter to determine the modulation of transcriptional complexes by PPARα activation at this site. Consistent with experiments presented in Figure 4, we confirmed that ligand-induced PPARα activation inhibits E2F-1 binding to the TERT promoter (Figure 5A). Inhibition of E2F-1 binding by PPARα ligands was dependent on the presence of PPARα and p16 because the efficacy of the ligands to repress E2F-1 binding was decreased on siRNA-mediated knockdown of either protein.
Because the trans-activation potential of E2F is repressed by the RB family of proteins,2 we next analyzed their occupancy at the E2F1 site in the TERT promoter. Treatment with Wy14,643 or FA resulted in the recruitment of the p107 and p130 proteins to the E2F1 site in control-transfected hVSMCs, an effect that was prevented by knockdown of PPARα or p16 (Figure 5B and 5C). In contrast, a modest interaction of pRB with the E2F1 site was not regulated by PPARα ligands (Figure 5D). Complementary results were obtained in murine coronary artery VSMCs, in which Wy14,643 induced the recruitment of p107 and p130 to the TERT promoter in wild-type cells but not in PPARα- or p16-deficient murine VSMCs (supplemental Figure II). Taken together, these data indicate that ligand-dependent activation of the PPARα/p16 pathway represses TERT transcription by recruiting p107 and p130 to the proximal TERT promoter, which alters the ability of E2F-1 to trans-activate the TERT promoter.
TERT Mediates the Growth-Inhibitory Effects of PPARα Activation in VSMCs
To investigate whether the inhibition of VSMC proliferation by PPARα is mediated by targeting TERT, we infected hVSMCs with adenoviral constructs coexpressing green fluorescent protein (GFP) and TERT (Ad-GFP/TERT) or overexpressing GFP alone (Ad-GFP) (Figure 6A). Infection of hVSMCs with 10 and 100 PFU per cell of Ad-GFP/TERT resulted in a significant increase of telomerase activity and cell proliferation. Consistent with our previous study,14 the PPARα ligand FA dose-dependently inhibited hVSMC proliferation in cells infected with control Ad-GFP. However, this efficacy of FA to inhibit hVSMC growth was either partially or completely inhibited in hVSMCs infected with 10 or 100 PFU per cell, respectively. Overexpression of TERT further prevented the ability of FA to arrest hVSMCs in G0/G1 phase and inhibit S phase entry (supplemental Figure III).14 Conversely, knockdown of TERT expression decreased telomerase activity and cell proliferation while preventing the growth-inhibitory effect of FA (Figure 6B). Collectively, these data demonstrate that activation of telomerase by overexpressing TERT induces VSMC proliferation and that TERT constitutes an important target for the inhibition of VSMC proliferation by PPARα.
PPARα Activation Inhibits Telomerase Activation During the Proliferative Response Underlying Neointima Formation In Vivo
To finally determine whether the downregulation of telomerase activity by PPARα ligands is applicable in vivo, we performed femoral artery denudation injuries in mice treated with FF (0.02%, 0.05%) or GF (0.5%). Using liver tissues from these mice, we verified that FF (0.05%) and GF (0.5%) significantly induced mRNA expression of the bona fide PPARα target gene acyl-CoA oxidase19 to a similar extent (supplemental Figure IV). As reported in previous studies,9,20 we observed a significant induction of telomerase activity 48 hours after injury (Figure 7A). Telomerase activation following injury was significantly decreased in mice treated with the PPARα agonists FF (FF 0.02%, 44.0±7.5% inhibition, P=0.02; FF 0.05%, 58.9±7.5% inhibition, P=0.003) and GF (GF 0.5%, 65.6±3.6% inhibition, P<0.001). The ability of PPARα to repress TERT expression during the proliferative response was further addressed using a carotid injury mouse model (Figure 7B).14 TERT mRNA levels were induced 24 hours after injury in carotid segments isolated from wild-type mice. This induction was augmented in tissues isolated from PPARα−/− mice and prevented by FF treatment in wild-type mice. Together, these data demonstrate that PPARα activation suppresses TERT expression and telomerase activation in response to vascular injury in vivo and point to an important role of TERT for the inhibition of neointimal VSMC proliferation by PPARα ligands.
Telomere maintenance is essential for cell proliferation and the primary mechanism preventing telomere attrition is through the action of telomerase.4 Earlier work has demonstrated that telomerase is activated in response to mitogenic stimulation of VSMCs and following vascular injury.8–10 Although these studies pointed to the control of telomerase activation as an important mechanism regulating VSMC proliferation,7,8 the transcriptional pathways underlying TERT expression and the mechanisms by which telomerase activation contributes to cell proliferation remain elusive. In the present study, both ectopic TERT expression and TERT siRNA approaches confirm a key role of telomerase activation in the control of VSMC proliferation and cell cycle progression. By exploiting the underlying transcriptional mechanisms governing TERT expression in VSMCs, we demonstrate that TERT transcription in VSMCs depends on a functional E2F site located at −174/−170 in the TERT promoter. Because E2F-1 is activated in the late G1 phase as a result of the dissociation from hyperphosphorylated pRB proteins,2 these results identify TERT as a bona fide E2F-1 target gene and provide a molecular basis for the induction of TERT in response to mitogens.8,9 Consistent with this notion, overexpression of E2F has recently been found to induce TERT transcription in somatic cells.17,18 Moreover, expression of the upstream tumor suppressor and CDKI p16, which attenuates VSMC proliferation and intimal hyperplasia as we previously demonstrated,14 impinges on TERT transcription and telomerase activation. Therefore, these observations support the concept that E2F released during cell cycle progression contributes to the activation of telomerase, which further augments cell proliferation during mitogenic stimulation.
The second key observation presented here demonstrates that PPARα activation in VSMCs inhibits TERT gene expression and telomerase activity in vitro and during the proliferative response underlying neointima formation in vivo. Overexpression of TERT in VSMCs reversed the antiproliferative efficacy of PPARα ligands placing the inhibition of telomerase by PPARα in a central position for the growth-inhibitory effect of fibrates.14 At the molecular level, the inhibition of TERT gene expression by ligand-activated PPARα involves the inhibition of E2F-1 binding and the assembly of the pocket proteins p107 and p130 at the proximal TERT promoter. In contrast, binding of hypophosphorylated pRB to the proximal E2F site was unaffected by the PPARα ligand, which is consistent with recent studies suggesting that p107 and p130, but not pRB, are involved in the trans-repression of E2F-responsive genes.21 Our studies further demonstrate that p16 is required for the recruitment of p107 and p130 to the TERT promoter and the inhibition of E2F-1 binding by PPARα ligands. Because p16 expression is trans-activated through direct PPARα binding to a degenerated DR1 PPRE,14 a model appears conceivable in which PPARα-mediated telomerase repression requires p16 as an upstream transcriptional target gene (Figure 8). Consistent with these data, recent studies suggest that gene repression at E2F sites through the recruitment of p107 and/or p130 mediates p16-induced growth arrest.22,23 Furthermore, in the context of VSMC proliferation, p107 and p130 have been implicated in mediating cell cycle arrest, whereas p130 overexpression inhibits injury-induced neointima formation.24,25 Considering this evidence and our observation that PPARα or p16 deficiency increase TERT expression and telomerase activity (supplemental Figure V), we infer that PPARα inhibits VSMC proliferation by repressing telomerase activity through a p16-dependent recruitment of p107 and p130 to the TERT promoter, an effect that will ultimately alter the ability of E2F-1 to trans-activate TERT transcription.
VSMC proliferation is regulated through a complex network of interconnected pathways and transcriptional repression of the p16/RB/E2F pathway may not be the only means by which PPARα mediates antiproliferative signals. The search for endogenous factors regulating VSMC proliferation has recently characterized several novel nodal proteins required for VSMC division and survival, including survivin1,26,27 and the PBEF (pre–B-cell colony-enhancing factor).28 Interestingly, survivin, a member of the mammalian “inhibitor of apoptosis” family, enhances telomerase activity in tumor cells by inducing TERT expression, is transcriptionally repressed by pRB/p130, and is induced by E2F-dependent trans-activation in fibroblasts.29,30 Considering this similarity of transcriptional regulation, PPARα may repress survivin transcription in VSMCs, which could ultimately alter telomerase activation through a yet to be defined molecular mechanism. Similarly, van der Veer and colleagues have recently reported that PBEF enhances VSMC survival and extends lifespan, whereas its antagonism induces premature senescence.28,31 Although, to date, a link between PBEF and telomerase in the regulation of cell proliferation has not been reported, it is intriguing to speculate that both survivin and PBEF may induce telomerase to enhance cell survival and promote VSMC proliferation, a process that could be transcriptionally repressed by PPARα.
Clinically, the cardiovascular benefit of fibrates was initially attributed to their metabolic efficacy to improve dyslipidemia; however, recent studies suggest pleiotropic vascular effects including the inhibition of neointima formation.12,14 This study extends these observations and outlines repression of telomerase as previously unrecognized mechanism by which PPARα activation inhibits vascular VSMC proliferation. Fibrates are often coadministered with statins and given that both drugs inhibit VSMC proliferation,14,32 combination therapy could be most effective in preventing cardiovascular complications driven by aberrant VSMC proliferation. Although statins have been reported to limit VSMC proliferation by inhibiting Rho,32 we have previously demonstrated that the pleiotropic antiinflammatory statin effects are mediated through PPARα.33 Considering these findings and the remarkable similarity between the pleiotropic effects of statins and fibrates, it is intriguing to suggest a mechanistic link between both classes of drugs and further studies are warranted to assess their potential additive or synergistic effects on VSMC proliferation and characterize the involved mechanisms.
We thank Violeta Arsenescu, MD, for technical assistance in the isolation of femoral tissues and Dr Carole Amant for performing carotid arterial injuries.
Sources of Funding
This work was supported, in part, by NIH grant RO1 HL084611 (to D.B.) and by American Diabetes Association Research award 1-06-RA-17 (to D.B.). F.G. was supported by a Fulbright Research Scholar grant and American Heart Association Postdoctoral Fellowship Grant 0725313B. T.N. and Y.Z. were supported by American Heart Association Fellowship Grants 0725620B and 0815514D.
Original received October 29, 2007; resubmission received August 26, 2008; revised resubmission received September 12, 2008; accepted September 17, 2008.
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