ADAMTS-7 Mediates Vascular Smooth Muscle Cell Migration and Neointima Formation in Balloon-Injured Rat Arteries
The migration of vascular smooth muscle cells (VSMCs) plays an essential role during the development of atherosclerosis and restenosis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases in VSMC migration. A recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), is capable of degrading vascular ECM proteins. Here, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. ADAMTS-7 protein accumulated preferentially in neointima of the carotid artery wall after balloon injury. In primary VSMCs, ADAMTS-7 level was enhanced by the proinflammatory cytokine tumor necrosis factor α and growth factor platelet-derived growth factor-BB. ADAMTS-7 overexpression greatly accelerated and small interfering RNA knockdown markedly retarded VSMC migration/invasion in vitro. In addition, luminal delivery of ADAMTS-7 adenovirus to carotid arteries exacerbated intimal thickening nearly sixfold 7 days after injury. Conversely, perivascular administration of ADAMTS-7 small interfering RNA but not scramble small interfering RNA to injured arteries attenuated intimal thickening by 50% at 14 days after injury. Furthermore, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels. Replenishing COMP circumvented the promigratory effect of ADAMTS-7 on VSMCs. Enforced expression of COMP significantly suppressed VSMC migration and neointima formation postinjury, which indicates that ADAMTS-7 facilitated intimal hyperplasia through degradation of inhibitory matrix protein COMP. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and postangioplasty restenosis.
Media-to-intima migration of vascular smooth muscle cells (VSMCs) is pivotal to intimal thickening in atherosclerosis, restenosis after coronary angioplasty, and late failure of vein grafting.1 Normally VSMCs are quiescent and are surrounded by and embedded in an extracellular matrix (ECM) scaffold that acts as a barrier to VSMC migration. ECM degradation and remodeling require the activation of extracellular proteases, which in turn facilitate VSMC migration.2 Previous studies have emphasized potential roles for the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP; the serine proteinases plasminogen activator and plasminogen; and the cysteine proteinases cathepsins K, L, and S during matrix remodeling and VSMC migration.3 However, the identity of the matrix-degrading proteinases during pathological vascular remodeling in vivo has remained the subject of speculation.
The recently identified metalloproteinase family of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) also degrade ECM. First identified in 1997, ADAMTS already showed strong biological relevance.4 For example, ADAMTS-1 has angioinhibitory activities and is crucial for the development and function of the urogenital system.5 However, the relevance of the ADAMTS family to cardiovascular disease remains largely unknown. Recently, Jönsson-Rylander et al reported that ADAMTS-1 might expedite atherogenesis by cleaving the ECM protein versican.6 The 227Pro polymorphism in ADAMTS-1 was associated with a nearly 2-fold increased risk of coronary heart disease event.7 ADAMTS-4 and -8 were also identified as inflammatory-regulated enzymes in macrophage-rich areas of human atherosclerotic plaques.8
Our previous study showed that recently discovered ADAMTS-7 can directly bind to and possibly degrade the ECM protein cartilage oligomeric matrix protein (COMP) in cartilage and has been implicated in the pathogenesis of arthritis.9 Once thought to be localized only in the musculoskeletal system, COMP is now recognized as a normal component of vascular ECM in humans and has been implicated in attachment and haptotaxis of VSMCs.10 COMP has also been found in human atherosclerotic and restenotic lesions, which suggests its potential importance during pathological ECM remodeling and VSMC migration.
To test the hypothesis that ADAMTS-7 participates in VSMC migration and neointima formation, we characterized the expression and function of ADAMTS-7 in a rat vascular balloon-injury model. We demonstrate for the first time that ADAMTS-7 facilitates VSMC migration and intimal thickening after vascular injury. Perivascular application of small interfering (si)RNA targeting ADAMTS-7 greatly ameliorated the intimal hyperplasia. These effects are mediated, at least in part, by ADAMTS-7–dependent COMP degradation.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Animal Artery Injury
Male Sprague–Dawley rats, weighing 210 to 230 g, were used in all experiments for the carotid artery injury model. All studies followed the guidelines of the Animal Care and Use Committee of Peking University. Briefly, rats were anesthetized by intraperitoneal injection of chloral hydrate (300 mg/kg). A balloon catheter of 1.5-mm diameter (Medtronic, Minneapolis, Minn) was introduced through the left external carotid artery and advanced 4 cm toward the thoracic aorta. The balloon was distended and then pulled back to the bifurcation with constant rotation. This procedure was repeated 2 more times to ensure complete endothelial denudation. Contralateral carotid arteries underwent a similar operation without injury and served as sham controls.
VSMC Culture and Real-Time Quantitative PCR Analysis
VSMCs were isolated from the thoracic aortic arteries of Sprague–Dawley rats (150 to 180 g) as described previously.11 Real-time PCR amplification involved use of an Mx3000 Multiplex Quantitative PCR System (Stratagene Corp, La Jolla, Calif) and SYBR Green I reagent and normalized to that of internal control β-actin.
Western Blot Analysis
Rat tissue extracts containing equal amounts of total protein (60 μg) were resolved by 10% SDS-PAGE. The membranes were incubated with primary antibody and IRDye 700DX-conjugated secondary antibody (Rockland Inc, Gilbertsville, Pa). The immunofluorescence signal was detected by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Neb).
Recombinant Adenovirus Construction
The recombinant adenovirus carrying nuclear factor (NF)-κB inhibitor subunit-α (IκB) was generously provided by Dr F. H. Bach (Harvard Medical School, Boston, Mass). The adenovirus expressing dominant-negative c-Jun (TAM67) and tTA gene were constructed as described previously.12 The adenoviruses for ADAMTS-7 or COMP were constructed and amplified according to the protocol of the manufacturer (BD Biosciences Clontech). An adenovirus carrying green fluorescence protein (GFP) was used as a negative control. For in vivo studies, a single exposure of 5×108 plaque forming units (pfu) of Ad-ADAMTS-7 or Ad-COMP adenovirus was luminally delivered to balloon-injured carotid segments and kept inside for 30 minutes to allow for sufficient infection. The adenovirus solution was subsequently removed and blood flow was restored.
ADAMTS-7 siRNA Transfection
ADAMTS-7 siRNA was designed by use of the Block-iT RNAi Designer and chemically modified by the manufacturer (Stealth siRNAs, Invitrogen). Sequences corresponding to the siRNA of ADAMTS-7 were: sense, 5′-CACAUCACCGUUGUGCGCCUUAUUA-3′; and antisense, 5′-UAAUAAGGCGCACAACGGUGAUGUG-3′. Transfection of rat VSMCs with the siRNA (50 nmol/L) in vitro was by use of Oligofectamine (Invitrogen). For in vivo studies, 15 μg of the siRNA dissolved in 30% pluronic gel solution was perivascularly delivered to the rat carotid arteries immediately after injury as described previously.13 A scramble Stealth RNAi duplex (catalog no. 12935, Invitrogen) served as a negative control. In a separate study, ADAMTS-7 or scramble Stealth siRNA was labeled with Alexa Fluor 555 (Invitrogen). The fluorescence signal was monitored by confocal laser scanning microscopy to demonstrate the efficiency of siRNA delivery to the carotid artery.
VSMC Migration and Invasion Assays
VSMC migration was assessed by scratch-wound assay as described previously.14 VSMC invasion analysis involved use of a modified Boyden chamber coated with an 8-μm barrier of reconstituted basement membrane protein similar in composition to that surrounding VSMCs in vivo (Chemicon International).15
Morphometric Analysis of Rat Carotid Arteries
Rat arteries were perfusion fixed with 4% paraformaldehyde. Briefly, cyrosections (7 μm thickness, 350 μm apart) were taken from the middle portion of the balloon-injured segment, and 8 slices of each sample were analyzed by hematoxylin/eosin staining and Spot Image software (Diagnostic Instruments, Australia). The intima and media areas and the circumference of the external elastic lamina were determined, and the ratio of intima to media areas was calculated.
All results are expressed as means±SEM. Statistical analysis involved use of the Student’s t test for comparison of 2 groups or 1-way ANOVA for multiple comparisons. P<0.05 was considered statistically significant.
Expression of ADAMTS-7 in Neointima of Injured Carotid Arteries
Mild to moderate intimal hyperplasia developed in balloon-injured rat carotid arteries at 7 days postinjury and became more severe at 14 days. Generally seen at low levels within normal vascular walls, ADAMTS-7 staining was markedly increased in the neointima in response to injury (Figure 1A). Dual immunofluorescence staining with specific antibodies against ADAMTS-7 or SMCs (SM α-actin) colocalized ADAMTS-7 predominantly to VSMCs in neointima (Figure 1B). Western blot analysis showed an initial decrease of ADAMTS-7 protein level in injured than in sham-operated arteries within the first 24 hours, then an increase during the 4 to 14 days after injury, with maximal expression at 7 days (Figure 1C).
ADAMTS-7 Expression Is Enhanced by Proinflammatory Cytokines and Growth Factor in VSMCs
Because ADAMTS-7 expression mainly localized to VSMCs in injured arteries, we examined the response of ADAMTS-7 to various stimuli relevant to vascular injury in primary cultured VSMCs. ADAMTS-7 mRNA and protein levels were induced by proinflammatory cytokines tumor necrosis factor (TNF)-α (25 μg/L), interleukin (IL)-1-β (40 μg/L), and growth factor platelet-derived growth factor (PDGF)-BB (10 μg/L) (Figure 2A and 2B). Elevation of ADAMTS-7 mRNA level by TNF-α was time- and concentration-dependent, beginning at 12 hours and peaking around 48 hours with 25 μg/L TNF-α (Figure 2C); maximal upregulation occurred with 50 μg/L TNF-α for 24 hours (Figure 2E). ADAMTS-7 protein level showed similar response to TNF-α (Figure 2D and 2F). By contrast, proatherosclerotic factors oxidized LDL (50 mg/L) and homocysteine (125 μmol/L) did not alter ADAMTS-7 mRNA and protein expression. Additionally, antiinflammatory factor TGF-β (5 μg/L) downregulated ADAMTS-7 expression by 50% that of controls (Figure 2A and 2B).
NF-κB and AP-1 Mediate TNF-α–Induced ADAMTS-7 in Primary VSMCs
Genome-wide analysis of the ADAMTS-7 promoter by use of the programs TESS and TFSEARCH predicted the existence of proinflammatory element binding sites, including transcription factor NF-κB and AP-1 sites, which allowed for characterizing the potential signal pathway mediating the TNF-α–stimulated transcriptional regulation of ADAMTS-7 (Figure I in the online data supplement). Application of an ectopic IκB adenovirus or c-Jun dominant-negative adenovirus TAM67 ameliorated the TNF-α–induced ADAMTS-7 elevation in VSMCs by 61% and 44%, respectively (Figure 2G and 2H), whereas synergistic application of the two viruses completely abolished TNF-α–mediated ADAMTS-7 induction (Figure 2I). In addition, ChIP-PCR analysis confirmed a bona fide interaction of NF-κB or AP-1 to the corresponding response elements within the ADAMTS-7 promoter (Figure 2J and supplemental Figure II). Thus, our data suggest that both NF-κB and AP-1 are critical for the TNF-α–induced ADAMTS-7 gene expression in VSMCs.
ADAMTS-7 Facilitates Migration of VSMCs
Infection of VSMCs with ADAMTS-7 adenovirus (Ad-ADAMTS-7) at 10 multiplicities of infection (mois) markedly increased ADAMTS-7 protein level compared with Ad-GFP–infected VSMCs (Figure 3A). Therefore, we used 10 mois in subsequent studies. In vitro scratch-wound assay revealed enhanced migration of Ad-ADAMTS-7–infected VSMCs compared with Ad-GFP–infected cells (Figure 3C). The mean migration distance was 3.5-, 2.4-, and 2.4-fold longer than that in Ad-GFP–infected cells at 6, 12, and 24 hours, respectively, after injury. Additionally, invasion assay with PDGF-BB used as a chemoattractant and recombinant basement membrane protein as a barrier showed approximately 4.3-fold more migration activity of Ad-ADAMTS-7–infected VSMCs than Ad-GFP–infected cells at 6 hours after injury (Figure 3D). By contrast, migration of cells through the barrier did not differ in the absence of ECM in the barrier (data not shown). Thus, ADAMTS-7 facilitates VSMC migration through degradation of ECM component.
ADAMTS-7 Exacerbates Neointimal Thickening
Ad-ADAMTS-7 was luminally delivered to injured vessel walls to assess the effect of ADAMTS-7 on VSMC migration in vivo. Figure 3B shows a representative Western blot from Ad-GFP– or Ad-ADAMTS-7–infected (5×108 pfu) arteries at 4 days after balloon injury (n=3 for each group). En face staining 4 days after balloon injury showed that Ad-ADAMTS-7 infection caused greater intimal cell accumulation than Ad-GFP infection (Figure 3E; 215±21 versus 92±15 per high-power field of intimal surface under light microscopy, P<0.05; n=3 for each group). The cells observed in the neointima at 4 days might be migrating rather than proliferating cells.16 At 7 days after injury, the neointima area of the vascular wall was 6-fold greater in Ad-ADAMTS-7–infected than in Ad-GFP–infected vessels (Figure 3F and supplemental Figure III, A; P<0.05; n=6). The ratio of neointima to media area was significantly elevated in Ad-ADAMTS-7–infected vessels (supplemental Figure III, C). However, no difference was seen in media area and circumference of external elastic lamina (supplemental Figure III, B and D).
ADAMTS-7 Knockdown by siRNA Retards VSMC Migration
Knockdown of ADAMTS-7 by specific siRNA was verified by real-time PCR and Western blot analysis (Figure 4A). Scratch-wound assay showed that ADAMTS-7 siRNA knockdown significantly reduced, by 50%, the mean migration distance of VSMCs as compared with scramble siRNA at 6 hour after injury (Figure 4B).
ADAMTS-7 Knockdown by siRNA Ameliorates Neointima Formation in Response to Injury In Vivo
We next examined whether ADAMTS-7 siRNA knockdown could reduce intimal hyperplasia in vessels in response to balloon injury in vivo. Pluronic gel containing scramble or ADAMTS-7 siRNA was perivascularly applied to injured arteries. To demonstrate the efficiency of siRNA delivery into the carotid artery by pluronic gel, both scramble and ADAMTS-7 Stealth siRNA were labeled with Alexa Fluor 555 (Invitrogen), and successful transfection was evidenced by red stains seen under confocal microscopy 7 days after injury (Figure 5A). No or weak red staining was seen without siRNA application. Accordingly, immunohistochemistry staining revealed reduced ADAMTS-7 expression and attenuated intimal thickening in the neointima area in Alexa Fluor–labeled ADAMTS-7 siRNA-treated arteries as compared with scramble siRNA-treated arteries (Figure 5A). Furthermore, Western blot analysis demonstrated that application of ADAMTS-7 siRNA potently inhibited ADAMTS-7 protein expression in the injured arteries as compared with scramble siRNA at 7, 10, and 14 days after injury (Figure 5B; n=4 to 6 per group). Consistent with this observation, the neointima area of arteries with ADAMTS-7 siRNA knockdown was reduced, by ≈70% that with scramble siRNA knockdown (1.00±0.48 versus 3.52±1.17×104 μm2; n=6, P<0.05; Figure 6A and 6B) at 7 days. Ten days after injury, the neointimal area of ADAMTS-7 siRNA– and scramble siRNA–treated arteries was 3.37±0.90 and 7.81±1.44×104 μm2, respectively (n=8 per group, P<0.05). Even 14 days after injury, the neointimal area with ADAMTS-7 siRNA knockdown was still almost half that with scramble siRNA treatment (6.47±2.30 versus 12.95±1.39×104 μm2; n=8 to 12 per group, P<0.05). Accordingly, the ratio of neointima to media area was significantly lower in ADAMTS-7 siRNA– than scramble siRNA–treated arteries from 7 to 14 days (Figure 6D). In contrast, the media area and circumference of external elastic lamina did not differ with the two treatments (Figure 6C and 6E).
ADAMTS-7 Facilitates VSMC Migration Through Degradation of COMP
Western blot analysis verified the existence of COMP (110 kDa) within the vessel wall (Figure 7A). With increased ADAMTS-7 protein level with injury, full-length COMP level decreased and COMP fragment (≈65 kDa) accumulated (n=3 to 5 per group, P<0.05), which indicating a parallel alteration between increased ADAMTS-7 level and COMP degradation in injured arteries. Previously by use of recombinant ADAMTS-7 and purified COMP protein, we have already demonstrated ADAMTS-7 directly binds to and degrades COMP in vitro.9 However, whether ADAMTS-7 also mediates COMP cleavage in native vessels or VSMC is still unclear. Because protein–protein interaction is fundamental process for most enzyme–substrate reaction, we included coimmunoprecipitation assay to verify the association of ADAMTS-7 and COMP in vivo. A specific COMP band was present in the immunoprecipitated complexes with anti–ADAMTS-7 but not control IgG antibodies, and vice versa (supplemental Figure IV), which demonstrates that ADAMTS-7 specifically binds to COMP in both native vessel walls and VSMCs. To further verify that degradation of COMP in the injured artery was attributable to ADAMTS-7, VSMCs were infected with increasing amount of ADAMTS-7 adenovirus. Overexpression of ADAMTS-7 in VSMCs led to a dose-dependent decrease of full-length COMP and parallel increase of COMP fragment (Figure 7B), similar to the in vivo observation. Furthermore, ADAMTS-7 overexpression led to decreased full-length COMP and increased COMP fragment in injured arteries (Figure 7C). In contrast, ADAMTS-7 knockdown resulted in less COMP cleavage and more full-length COMP accumulation (Figure 7D). These data strongly suggest a causal role of ADAMTS-7 on COMP degradation in VSMCs and injured vessels.
To further test the hypothesis that ADAMTS-7 facilitated VSMC migration and neointima formation through degradation of vascular COMP, VSMCs were replenished with excessive COMP by adenoviral infection (supplemental Figure V, A). COMP overexpression significantly attenuated ADAMTS-7 facilitated VSMC migration (Figure 8A). Concomitantly, both scratch-wound (Figure 8B) and Boyden chamber (Figure 8C) assay demonstrated that COMP itself significantly inhibited migration activity of VSMCs. To confirm the inhibitory effect of COMP in vivo, COMP adenovirus was expressed in the injured arteries (supplemental Figure V, B). COMP indeed manifested pronounced suppressive effect of the neointima formation. The neointima area of Ad-COMP–treated injured arteries were 63% (day 7) and 37% (day 14) less than that of Ad-GFP–treated arteries (Figure 8D and 8E). Collectively, these results suggest that ADAMTS-7 facilitates VSMC migration and thus contribute to the development of neointimal lesions through COMP degradation.
Our in vitro and in vivo results demonstrate a novel role for ADAMTS-7 in VSMC migration and intimal hyperplasia in response to vessel injury. ADAMTS-7 expression was induced by balloon injury in rat arteries and TNF-α or PDGF-BB stimulation in primary VSMCs. ADAMTS-7 upregulation greatly accelerated VSMC migration/invasion and contributed to neointimal thickening in injured arteries. We also show that in vivo delivery of ADAMTS-7 siRNA inhibited neointima formation. Furthermore, we reveal that the promigratory effect of ADAMTS-7 depends, at least in part, on degradation of the ECM factor COMP.
The early induction of the enzymatic activity of ECM-degrading proteases precedes the appearance of VSMCs in neointima.17 From the therapeutic standpoint, inhibition of such proteases would be beneficial for both atherosclerosis and restenosis. Given the disappointment that broad-spectrum protease inhibitors (such as broad-spectrum MMP inhibitors) have led to severe side effects in clinical trials, one would speculate that more selective protease inhibitors are needed. Thus, unraveling the role of individual proteases in cardiovascular disease is of particular importance. To the best of our knowledge, this study is the first to show the functional importance of ADAMTS-7 in VSMC migration and neointima formation in response to injury and thus ADAMTS-7 may serve as a novel therapeutic target for vascular disease. ADAMTS-7 expression was induced by balloon injury in arteries and by proinflammatory cytokines IL-1, TNF-α, growth factor PDGF-BB, and reactive oxygen species H2O2 stimulation in primary VSMCs. The above stimuli have all been involved in vascular injury and may account for the induction of ADAMTS-7 in the injured arteries in vivo.11,16,18 Consistent with our observation, ADAMTS-7 mRNA was previously reported to be upregulated by TNF-α in the human monocyte/macrophage cell line THP-1,8 but the underlying regulatory mechanism was not elucidated. Here, we describe the potential signaling pathway mediated through transcriptional factors NF-κB and AP-1 by which TNF-α may stimulate ADAMTS-7 induction in rat VSMCs. Our results expand early studies that proved the contribution of AP-1 and NF-κB in TNF-α induced MMP-9 activation,19 which is also essential for VSMC proliferation and migration into the intima.
Another key finding of the present study is identification of the potential mechanism of the promigratory effect of ADAMTS-7 on VSMC migration and subsequent neointima formation. To date, substantial evidence has supported the key role of ECM dysfunction, namely excessive synthesis or degradation of ECM, during pathological states such as atherosclerosis and restenosis. Abnormal turnover of vascular ECM components such as versican and collagen contribute to the progression of vascular diseases.20 In this study, we first identified the degradation of a normal ECM protein COMP within injured arteries (Figure 7A). The size of the degraded fragments of COMP within injured vessels greatly resembles that from arthritic cartilage,21 which suggests a common regulatory mechanism of COMP catabolism in both cartilage and vessels. Indeed, we verified that the existence of COMP fragments in injured arteries is attributable, at least in part, to elevated ADAMTS-7 level by manipulating ADAMTS-7 with adenovirus or siRNA (Figure 7B through 7D). Furthermore, we observed that replenishing COMP reversed the ADAMTS-7–facilitated VSMC migration, which indicates the importance of ADAMTS-7–mediated COMP degradation during this process. Previously, Riessen et al showed COMP possibly playing a role in the attachment and haptotaxis of VSMCs.10 Early studies also implicated COMP in maintaining ECM homeostasis through binding to other ECM components such as collagen I, II, and IX and matrilin.22 A recent study by Halász et al further showed COMP as a catalyst in collagen fibrillogenesis.23 In our study, we observed a novel inhibitory effect of COMP on VSMC migration and neointima formation, although the underlying mechanism needs to be further clarified. Collectively, ADAMTS-7–mediated excessive degradation of an inhibitory protein COMP in vessels may break the delicate balance of ECM homeostasis and contribute to the migration of VSMCs and intimal hyperplasia.
In our study, sustained in vivo knockdown of ADAMTS-7 was achieved through delivering a chemically modified siRNA by use of pluronic gel to the rat carotid artery. RNA interference is emerging as an extremely potent and promising therapeutic strategy to suppress gene expression and is now being evaluated in clinical trials. Many studies have demonstrated the power of in vivo administration of RNA interference in mammals,24 and the stability of siRNA, route of administration, and choice of delivery tool are crucial for success. Pluronic gel has been used successfully for local perivascular delivery of antisense oligonucleotides, natriuretic peptide, rapamycin, suramin, or other substances in experimental carotid angioplasty.13,25,26 However, perivascular application of siRNA with pluronic gel has not been tested before. With our use of this strategy, intimal hyperplasia was substantially retarded 7 days after balloon injury, and the inhibitory effect was sustained for up to 2 weeks. The effectiveness was possibly achieved by sustained release of siRNA from the pluronic gel, the increased stability of chemically modified siRNA,27 and the ease of the siRNA duplex penetrating the loose connective tissue of tunica adventitia. The prolonged success with “naked” siRNA duplexes applied to tissues has been reported in the eye, lung, heart, carotid artery, and central nervous system, although the underlying mechanism remains largely unknown.28,29 Our demonstration that ADAMTS-7 silencing may prevent neointima formation highlights the potential of targeting ADAMTS-7 by siRNA as an applicable therapeutic tool to ameliorate intimal hyperplasia.
In conclusion, our studies demonstrate a novel mechanism of vascular ECM remodeling and VSMC migration. ADAMTS-7 and ADAMTS-7–mediated COMP degradation may play an important role in human cardiovascular disease states such as atherosclerosis and restenosis after angioplasty.
We thank Li Chen, Qiang Shen, and Jing Li from the Peking University Health Science Center for excellent technical assistance.
Sources of Funding
This work was supported by National Natural Science Foundation of the People’s Republic of China grants 30670849 (to W.K.) and 30821001 (X.W.), Natural Science Foundation of Beijing grant 7072039 (to W.K.), Program from the Ministry of Education of China “New Century Excellent Talents in Universities” (to W.K.), National Basic Research Program of the People’s Republic of China grant 2006CB503802 (to Q.X.), and the Chang Jiang Scholars Program (to Q.X.).
Original received September 28, 2008; revision received January 8, 2009; accepted January 12, 2009.
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