A Novel Loss-of-Function DDAH1 Promoter Polymorphism Is Associated With Increased Susceptibility to Thrombosis Stroke and Coronary Heart Disease
Rationale: Asymmetrical dimethylarginine (ADMA), an endogenous arginine analogue, inhibits nitric oxide synthases and plays an important role in endothelial dysfunction.
Objective: In the present study, we tested whether a novel genetic variant in dimethylarginine dimethylaminohydrolase 1 (DDAH1), an important ADMA hydrolyzing gene, was associated with stroke and coronary heart disease (CHD) susceptibility in the Chinese Han population.
Methods and Results: By resequencing, we identified a novel 4-nucleotide deletion/insertion variant in the DDAH1 promoter. The insertion allele disrupted binding of metal-regulatory transcription factor 1, which resulted in significant reduction of in vitro DDAH1 transcriptional activity and in vivo DDAH1 mRNA level, and in turn, increased plasma ADMA level and the ratio of ADMA to l-arginine. We initially genotyped the polymorphism in 1388 stroke patients and 1027 controls as well as 576 CHD patients and 557 controls and then replicated our study in additional independent case-control cohorts comprising 961 stroke patients and 822 controls and 482 CHD patients and 1072 controls. We identified that the −396 4N ins allele was significantly associated with increased risk of thrombosis stroke and CHD after adjusting for environmental factors in both samples for both diseases (thrombosis stroke discovery set: odds ratio [OR]=1.35, P=0.032; replication set: OR=1.51, P=0.006; CHD discovery set: OR=1.45, P=0.035; replication set: OR=1.47, P=0.003).
Conclusions: Our results suggest that the DDAH1 loss-of-function polymorphism is associated with both increased risk of thrombosis stroke and CHD.
Nitric oxide (NO) is synthesized from l-arginine by 3 different isoforms of NO synthases (NOS), neuronal NOS, endothelial NOS (eNOS), and inducible NOS. It plays important roles in the maintenance of normal vascular homeostasis1 and protection of blood vessels from injury caused by atherogenic processes such as smooth muscle cell proliferation, platelet aggregation, monocyte adhesion, and oxidative modification of low-density protein.2 The synthesis of NO is inhibited by the endogenous arginine analogue, asymmetrical dimethylarginine (ADMA).3 Elevations in plasma ADMA result in reduced NO generation, which has been observed in numerous disorders including stroke,4 atherosclerosis,5,6 hypertension,7,8 hyperlipidemia,9,10 diabetes mellitus,11,12 and renal failure. Moreover, ADMA level is associated with acute cardiovascular events and death.13,14
The major pathway for ADMA clearance is hydrolysis by dimethylarginine dimethylaminohydrolase (DDAH) to l-citrulline and dimethylamines.15 To date, 2 distinct human DDAH isoforms (DDAH1 and DDAH2) have been identified, which overlap in tissue distribution.16 DDAH2 contributes only a small amount to the overall DDAH activity in many tissues, whereas interruption of DDAH1 leads to accumulation of ADMA and reduction in NO signaling.17 Therefore, we speculate that DDAH1 is the key regulator of ADMA metabolism. Because of its important role in the regulation of NO synthesis via modulating endogenous ADMA levels, DDAH1 is a potential candidate gene for risk of cardiovascular diseases; however, the association of this gene with atherosclerotic diseases, such as stroke and coronary heart diseases (CHD), has not, to our knowledge, been previously examined. In the present study, we resequenced the DDAH1 gene to identify all putative functional variants, including a novel 4-nucleotide deletion–insertion polymorphism (−396 4N del/ins) in the DDAH1 promoter. We performed functional analyses of this variant, determining that the mechanism by which it affects gene expression is via disruption of a metal-regulatory transcription factor (MTF)1 binding site and investigated whether this promoter variant was associated with stroke and CHD in the Han Chinese population.
Recruitment for the Discovery and Replication Samples
We carried out parallel genetic association studies for stroke and CHD in independent “discovery” and “replication” samples for each disease outcome. Details on sample recruitment, inclusion criteria, data collection, definition of risk factors and statistical power considerations are provided in the expanded Methods section, available in the Online Data Supplement at http://circres.ahajournals.org. In brief, the stroke discovery sample included 1388 CT- or MRI-confirmed stroke patients recruited from hospitals in Wuhan, China, and 1027 healthy, community-based controls. As described below, 48 of the stroke discovery sample controls were randomly selected for genetic variation screening, and (a different set of) 44 of these controls were selected based on genotype and risk factors for in vivo transcription and ADMA/NOS metabolite assays. The stroke replication sample (previously described elsewhere18) included 961 stroke patients and 822 controls. The CHD discovery sample (576 cases and 557 controls) and replication sample (482 cases and 1072 controls) were both comprised of patients treated at Tongii hospital in Wuhan, China, and disease-free, community-based controls. All patients and controls were carefully matched by geographic region of recruitment, were of Han Chinese ancestry, and provided written informed consent. This study was approved by the institutional ethics committees of the local participating hospitals.
Genetic Variation Screening
Common polymorphisms in DDAH1 (GenBank accession no. NC_000001) were identified by direct sequencing of genomic DNA derived from 48 randomly selected individuals from the stoke control sample. PCR arrays were designed to amplify regions up to 1.0 kb upstream from transcription-initiation sites (ie, the putative proximal promoter region), all exons, and adjacent noncoding regions. Details regarding primers and resequencing procedures are given in the Online Data Supplement.
The novel DDAH1 promoter polymorphism was genotyped using the TaqMan 5′-nuclease assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, Calif) under standard conditions. Details regarding primers and probes as well as TaqMan 5′-nuclease assay procedures can be found in the Online Data Supplement.
In Vitro Promoter–Variant Vector Construction
To determine whether the −396 4N deletion–insertion polymorphism influences transcription of the DDAH1 gene, we constructed 2 reporter plasmids encompassing −578 to +243 bp of the human DDAH1 promoter by PCR from 2 genomic DNA samples with the −396 4N del/del and −396 4N ins/ins genotypes, amplified using forward primer 5′-GCGCAGATCTGACCCGTGGGATTTAAGCTCAAC-3′ and reverse primer 5′-GCGCAAGCTTGGCCACGTCCTCCACGAAGA-3′. The PCR product was digested with BglII and HindIII and ligated into a pGL3-Basic vector (Promega, Madison, Wis) upstream of the reporter gene. All constructs used in this study were restriction mapped and sequenced to confirm their authenticity. The constructs were designated pGL3–4N-del and pGL3–4N-ins.
DDAH1 Promoter Analysis In Silico
The search for binding sites of potential transcriptional factors in the promoter region of DDAH1 was performed online at Genomatix with the MatInspector program.19
Cell Culture, Transient Transfection, and Luciferase Activity Assays
Cell culture and transient transfection procedures used in this study are fully described in the Online Data Supplement. Cells were harvested 36 to 48 hour after transfection using the reporter lysis buffer (Promega). Firefly and Renilla luciferase activity was analyzed at room temperature in a chemiluminometer (GloMax, Promega) according to the manufacturers’ instructions using the Dual-Luciferase Reporter Assay System (Promega). For each experiment, relative luciferase activity was defined as the mean value of the firefly luciferase/Renilla luciferase ratios obtained from 3 independent experiments. The (2-tailed) t test was used to compare expression levels of pGL3–4N-del and pGL3–4N-ins constructs.
Electrophoretic Mobility-Shift Assay
Details of the electrophoretic mobility-shift assay performed in this study are described in the Online Data Supplement.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation assays were performed using commercially available assay kits. Detailed descriptions are available in the Online Data Supplement.
Transcription Assays of the DDAH1 Gene
Transcription assays and ADMA/NOS metabolite assays (described below) were performed in 44 subjects from the stroke discovery sample controls. Twenty-two subjects homozygous for the −396 4N del allele were matched to an equal number of subjects with one or 2 copies of the −394 4N ins allele (16 heterozygotes and 6 homozygous) based on sex, age (within 1 year), and BMI (within 1 kg/m2). These subjects were not comorbid for other conventional risk factors; all had normal blood pressure (<140/90 mm Hg and no use of antihypertensive drugs), were nondiabetic (fasting glucose <7.8 mmol/L), and had normal lipid profiles.
RNA isolation, mRNA transcription and PCR performed in this study are described in the Online Data Supplement. Absolute quantification methods were used to measure DDAH1 and GAPDH mRNA levels. Assays for each subject were carried out in triplicate. Expression of DDAH1 relative to GAPDH (ie, DDAH1 copies per 1000 GAPDH copies) was compared between individuals with and without one or more copies of the −396 4N ins allele via the (2-tailed) paired t test.
ADMA/NOS Pathway Metabolite Assay
To investigate whether the −396 4N deletion–insertion polymorphism influences plasma ADMA and related endogenous metabolites, plasma concentrations of ADMA, symmetrical dimethylarginine (SDMA), and l-arginine were analyzed by high-performance liquid chromatography with fluorescence detection according to the modified method described by Teerlink et al.20 Details for this method are provided in the Online Data Supplement. Test subjects (n=44; ie, the same subjects included in transcription assays, above) were selected from the stroke control group, including 22 individuals homozygous for the −396 4N del allele, and 22 individuals with one or both copies of the −396 4N ins allele (16 heterozygous and 6 homozygous). The potential confounding effects of risk factors on ADMA serum levels were avoided by matching subjects on conventional risk factors as described above. Intra- and interassay coefficients of variation for all analyses were <1.2 and <3%, respectively. For each participant the ratio of plasma ADMA to l-arginine was calculated. ADMA, SDMA, and l-arginine levels were measured as the means of triplicate assays performed for each subject. Metabolite levels were compared between individuals with and without one or more copies of the −396 4N ins allele via the (2-tailed) paired t test.
Genetic Association of DDAH1 With Stroke and CHD
Baseline characteristics for stroke and CHD cases and controls were compared by two-tailed t test (for quantitative variables) and χ2 test (for qualitative variables). Assuming a dominant genetic model (chosen based on results of functional analyses and genotype frequencies), logistic regression was used to test for genetic association in stroke and CHD samples with and without adjusting for sex, age, body mass index, hypertension, diabetes, hyperlipidemia and smoking status. For all statistical tests, we used a threshold of α=0.05 for statistical significance.
Statistical analyses were performed in SPSS 13.0 (SPSS Inc, Chicago) for Windows (Microsoft Corp, Redmond, Wash) and SNPassoc for the R statistical package.21 Linkage disequilibrium among DDAH1 polymorphisms was indicated by D′. Deviations from Hardy-Weinberg equilibrium were assessed by χ2 test using Haploview 4.0.22
Identification of Polymorphisms in the DDAH1 Gene
By resequencing the DDAH1 gene in 48 randomly selected unrelated individuals from the Han Chinese population, we identified 9 polymorphisms, including one in the promoter region, one in exon 4, five in intronic regions, and 2 in a 3′ untranslated region (Table 1). All polymorphisms were in Hardy–Weinberg equilibrium in our sample. The coding variant in exon 4, which was a synonymous single-nucleotide polymorphism (SNP) and therefore unlikely to be of clinical importance, was not further investigated in this study. Likewise, the intronic and 3′ untranslated region SNPs, which were not located within potential AU-rich elements or micro-RNA target sequences (based on bioinformatics analysis), were not further studied. However, a novel four-nucleotide deletion–insertion polymorphism (−396 GCGT→del) in the DDAH1 promoter region was identified, which was in low linkage disequilibrium with the 8 other identified SNPs (Online Figure I). The homozygous −396 4N ins/ins genotype and the heterozygous −396 4N del/ins genotype were observed in 2.1% and 16.7% of the sample, respectively. Given the frequency of the −396 4N ins allele (ie, 10 of the 96 alleles; 10.4%) and its potential to affect transcription activity of the DDAH1 gene, further functional studies of this polymorphism were performed.
DDAH1 −396 4N Deletion–Insertion Polymorphism Affects Transcriptional Activity
To test whether the −396 4N deletion–insertion polymorphism in the DDAH1 promoter region was functionally important for the regulation of DDAH1 transcription, we performed functional analyses comparing the activities of −396 4N del and −396 4N ins alleles in human umbilical vein endothelial cells (HUVECs), HEK293T, and HepG2 cells. HUVECs (endothelial) were chosen for their biological relevance to stroke and CHD, whereas HEK293T (kidney) and HepG2 (liver) cells were chosen because DDAH1 is known to be expressed primarily in kidney and liver tissues. As shown in Figure 1A, reporter gene expression for the −396 4N ins allele was significantly reduced compared to the −396 4N del allele (38.7±8.4% reduction in HUVECs, P<0.05; 78±3.7% reduction in HEK293T, P<0.001; and 87.5±2.7% reduction in HepG2, P<0.001). This evidence suggests that the 4-nucleotide insertion in the DDAH1 promoter region may affect a transcriptional regulator element binding domain that in turn affects gene expression.
DDAH1 −396 4N Deletion–Insertion Polymorphism Affects MTF1-Stimulated Transcription
Bioinformatics analysis suggested that the −396 4N ins allele destroys MTF1 binding site in the DDAH1 promoter. We then experimentally assessed the function of the DDAH1 −396 4N deletion–insertion polymorphism on MTF1-mediated gene transcription, using −396 4N del and −396 4N ins promoter reporter plasmids in HUVECs, HEK293T and HepG2 cells cotransfected with MTF1 expressing plasmids. As shown in Figure 1B, transfection of the MTF1 expressing plasmids activated transcription for the −396 4N del but not the −396 4N ins DDAH1 promoter construct in a dose-dependent manner. This indicates that the MTF1 binding site in the DDAH1 promoter was functional and that its disruption by the −396 4N ins allele caused a reduction in transcriptional activity of the gene.
MTF1 Binding Analysis for the DDAH1 −396 4N Del Allele
We performed DNA binding analysis in HUVECs, HEK293T and HepG2 cells to further assess the functional relevance of the −396 4N deletion–insertion polymorphism. As shown by electrophoretic mobility-shift assays, MTF1-containing nuclear extracts specifically bound to the biotin-labeled −396 4N del probe but not to the −396 4N ins probe (Figure 2), and additional unlabeled oligonucleotide partially competed with this binding. Moreover, preincubation with specific MTF1 antibodies supershifted the MTF1 band, confirming the specificity of the MTF1-DNA binding (Figure 2).
To determine whether the MTF1 proteins bound to the DDAH1 promoter in intact cells, we examined HUVECs, HEK293T, and HepG2 cells using the chromatin immunoprecipitation assay.23 As shown in Figure 3 (left), MTF1 binding to the −396 region of the DDAH1 promoter was detected in all of these cell types. We also performed this assay on lymphocytes isolated from 2 control subjects (from the stroke discovery sample) with −396 4N del/del and −396 4N ins/ins genotypes, respectively. As shown in Figure 3 (right), MTF1 bound to this region in the −396 4N del/del subject, but not the −396 4N ins/ins subject.
In Vivo Effects of the DDAH1 −396 4N Deletion–Insertion Polymorphism on mRNA Levels
Taken together, the results of the in vitro experiments, above, suggest that the −396 4N ins allele disrupts binding of the MTF1 complex to the DDAH1 promoter, which, in turn, reduces transcription efficiency. To confirm the influence of this polymorphism on DDAH1 transcription activity in vivo, we compared DDAH1 mRNA relative expression in lymphocytes of 44 matched control subjects (from the stroke discovery sample), including 22 subjects homozygous for the −396 4N del allele and an equal number of subjects with one or more copies of the −396 4N ins allele (ie, 6 homozygotes and 16 heterozygous). DDAH1 and GAPDH mRNA levels were quantified by TaqMan real-time quantitative PCR analysis. As shown in Figure 4, DDAH1 expression in lymphocytes was reduced in subjects with one or both copies of the −396 4N ins variant (32.35±11.63 copies per 1000 GAPDH), compared to subjects homozygous for the −396 4N del allele (54.10±22.34 copies per 1000 GAPDH; P<0.001).
Association of DDAH1 −396 4N Deletion–Insertion Polymorphism With ADMA Levels
To further investigate the functional role of the −396 4N deletion–insertion polymorphism, we measured plasma concentrations of the ADMA/NOS pathway metabolite products in these 44 individuals. Median ADMA plasma concentrations were significantly higher in individuals with one or both copies of the −396 4N ins allele (0.70±0.26 μmol/L) compared to individuals homozygous for the −396 4N del allele (0.50±0.10μmol/L; P=0.002; Figure 5). Likewise, the ratio of ADMA to l-arginine was also increased in subjects with one or both copies of the −396 4N ins allele (P=0.003). In contrast, the plasma concentrations of symmetrical dimethylarginine (SDMA) and l-arginine did not differ by genotype (results not shown).
Association of DDAH1 −396 4N Deletion–Insertion Polymorphism With Thrombosis Stroke and CHD
Given the evidence from functional analyses of its effects on DDAH1 expression and plasma ADMA concentration, we tested the association of the −396 4N deletion–insertion polymorphism with CT- or MRI-documented stroke (discovery sample: 1388 cases and 1027 controls; replication sample: 961 cases and 822 controls) and with CHD (discovery sample: 576 cases and 557 controls; replication sample: 482 cases and 1072 controls). Baseline characteristics of stroke and CHD cases and controls from independent discovery and replication samples are shown in Online Tables I and II, respectively. The distributions of DDAH1 genotypes are shown in Online Tables III and IV. No deviations from Hardy-Weinberg equilibrium were observed in cases or controls from any of our four samples. The −396 4N ins allele was significantly associated with increased risk of thrombosis stroke in discovery and replication samples, both with or without adjustment for conventional risk factors including sex age, body mass index, hypertension, diabetes, hyperlipidemia, and smoking status (discovery sample: unadjusted odds ratio [OR]=1.33, P=0.017, adjusted OR=1.35, P=0.032; replication sample: unadjusted odds ratio OR=1.37, P=0.017, adjusted OR=1.51, P=0.006) (Table 2). In contrast, the significant genetic associations observed in the discovery sample for overall stroke (ie, all subtypes combined) and with lacunar and hemorrhage stroke subtypes, were not confirmed in the replication sample (see Online Tables III and IV). However, the −396 4N ins allele was significantly associated with increased risk of CHD, with or without adjustment for covariates, in both discovery and replication CHD samples (discovery sample: unadjusted OR=1.50, P=0.006, adjusted OR=1.45, P=0.035; replication sample: unadjusted OR=1.31, P=0.035, adjusted OR=1.47, P=0.003) (Table 2).
We have demonstrated that risks of stroke and CHD are associated with the novel −396 4N deletion–insertion polymorphism in the promoter region of the DDAH1 gene. The significant association remained even after adjustment for conventional vascular risk factors (ie, age, gender, hypertension, diabetes, hyperlipidemia and smoking status), suggesting that the contribution of this polymorphism to the risk of both stroke and CHD is independent of conventional vascular risk factors. Functional analyses showed that the mechanism by which transcriptional activity of the DDAH1 gene is reduced involves the disruption of a MTF1 transcription factor binding site by the −396 4N ins allele. Moreover, analysis of human samples showed that individuals with the −396 4N ins variant had significantly higher plasma ADMA level and ADMA to l-arginine ratio than those with 2 copies of the −396 4N del allele. Together, these findings support a hypothesis that genetic variants affecting DDAH1 gene expression modify stroke and CHD susceptibility through the ADMA/NOS pathway.
Specifically, the higher stroke and CHD risk in individuals with −396 4N ins variant could be attributable to the disruption of DDAH1 promoter activity causing down regulation of the DDAH1 gene, which, in turn, increases the plasma ADMA level and ADMA to l-arginine ratio. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation or reduced NO metabolite levels.24 Mounting evidence indicates that ADMA is a key player in endothelial dysfunction and is involved in the pathophysiology of a variety of cardiovascular diseases.5,6,10 ADMA arises from the degradation of proteins containing methylated arginine residues and is eliminated largely through active metabolism by DDAH. Therefore, DDAH dysfunction may lead to local accumulation or release of intracellular ADMA and thereby inhibition of eNOS in pathophysiological states.
According to studies in several different populations,14,25,26 even moderate increases in plasma ADMA level are associated with deterioration of endothelial function, and hence, associated with risk of vascular events. It has also been well established that disruption of methylarginine metabolism impairs vascular homeostasis.17 In addition, Bode-Boger et al reported that endothelial function could be improved by oral supplementation with l-arginine. This is probably attributable to the normalization of the ratio of ADMA to l-arginine,27 which is an indicator of eNOS impairment. However, little was previously known about genetic variants (especially those causing functional deficiency) in DDAH and their roles in human ADMA metabolism, eNOS impairment, and atherosclerotic disease susceptibility.
There are several limitations of our study that must be acknowledged. First, we have focused our attention on the −396 4N deletion–insertion polymorphism based on its potential biological function; however, other variants in DDAH1 or nearby genes in strict linkage disequilibrium with this polymorphism could be responsible for the observed genetic association. In the HapMap Han Chinese population (CHB), there was very low LD in the region surrounding the −396 4N insertion-deletion polymorphism, with the nearest LD blocks occurring ≈9 kb upstream and 36 kb downstream of this variant. On the other hand, in the HapMap CEU population (European ancestry), this polymorphism was located in a 45 kb block of fairly strong LD, which extended from 9kb upstream of the DDAH1 gene.28 Nevertheless, corroborating functional analyses showing the effect of this polymorphism on a plausible biological pathway contributing to vascular disease indicate that the −396 4N deletion–insertion polymorphism is likely to be the causal locus. Other limitations include the possibility of cryptic population substructure which could lead to a false-positive association. However, given the homogenous study population we expect population substructure to be minimal, and if present, to have negligible impact on our hypothesis-driven test for genetic association (in contrast to large-scale hypothesis-generating genetic association studies where population structure may cause false signals among ancestry-informative loci). Finally, the subjects recruited in our study may not be entirely representative of the general Han Chinese population; however, because we used a large number of subjects and replicated our findings in independent samples, our results are unlikely to be attributable to a specific cohort effect. Nevertheless, it is important to confirm these findings in prospective cohort studies.
Our study provides strong evidence that the novel −396 4N deletion–insertion polymorphism in DDAH1 is associated with both thrombosis stroke and CHD. Moreover, the high odds ratios (OR=1.35 to 1.51 and 1.45 to 1.47 for thrombosis stroke and CHD, respectively) and high frequency of the risk allele (ie, ≥10% in our stroke and CHD case/control samples) indicate that a substantial proportion of disease may be attributable to this risk locus. In conclusion, this study further supports the important role of ADMA in endothelial dysfunction and cardiovascular diseases, and brings to light a novel risk locus that may lead to better risk assessment and/or intervention for stroke and CHD.
We thank Drs Luo Zhang, Tao Zhang, Rui Li, Baozhen Tan, Ling You, and Yali Zhen for recruiting patients for this study and Qi Jiang for thoughtful advice and technical assistance in the genotyping work. We also acknowledge 2 anonymous reviewers for their thoughtful suggestions for improving this study.
Sources of Funding
This work was supported by grants from National Nature Science Foundation Committee of China (no. 30800458), National “863” project (no. 2006AA02A406), and “973” projects (nos. 2007CB512004 and 2006CB503801).
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Novelty and Significance
What Is Known?
Nitric oxide (NO), a signaling molecule involved in vascular homeostasis, is protective against atherosclerosis and blood vessel inflammation.
Plasma asymmetrical dimethylarginine (ADMA) inhibits the biosynthesis of NO and is elevated in some patients with cardiovascular diseases.
ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH).
What New Information Does This Article Contribute?
We discovered a novel loss-of-function insertion–deletion polymorphism in the promoter of the DDAH1 gene in the Han Chinese population.
We show that this polymorphism disrupts a key transcription binding site, causing reduction in DDAH expression.
We show that this polymorphism is associated with thromboembolic stroke and coronary heart disease in 2 independent Han Chinese samples.
The importance of genetics on cardiovascular diseases is well known; however, the pathways leading to disease, and the specific genes involved, are not fully understood. One candidate gene that may affect cardiovascular disease pathogenesis through its role in NO biosynthesis is DDAH1. We identified a novel loss-of-function polymorphism in DDAH1 that causes a reduction in gene expression, affects plasma concentrations of NO pathway metabolites, and increases risk of both thromboembolic stroke and coronary heart disease in Han Chinese. Our study confirms the importance of the NO pathway in vascular homeostasis and specifically implicates DDAH1 in disease pathogenesis. This discovery, and the incorporation of genetic information into risk models, may lead to better disease predictions. More generally, better understanding of the role of DDAH1 in the NO pathway may lead to the development of new biological targets and/or new therapeutic strategies for cardiovascular disease.
↵*These authors contributed equally to this work.
Original received July 27, 2009; resubmission received December 22, 2009; revised resubmission received January 29, 2010; accepted February 1, 2010.