ACE I/D and eNOS E298D gene polymorphisms in Saudi subjects with hypertension

Abstract

Background:

Hypertension has a multifactorial background based on genetic and environmental interactive factors.

Objectives:

We aimed to test for the association of the angiotensin-converting enzyme (ACE) and endothelial nitric oxide synthase (eNOS) gene polymorphisms with hypertension.

Subjects and methods:

Participants included 120 Saudi patients with hypertension and 250 normal healthy controls. For all participants, DNA was processed for characterization of ACE I/D and eNOS E298D gene polymorphisms.

Results:

Hypertensive cases showed a significantly higher frequency of the ACE mutant D allele carriage (98.3% vs. 92.4%, p = 0.028, OR = 4.8). Cases with hypertension associated with diabetes and obesity showed 100% mutant D allele carriage. Regarding the eNOS E298D polymorphism, the frequency of the mutant D allele carriage was only observed to be higher among cases with hypertension associated with diabetes and obesity, in comparison with controls, yet not reaching statistical significance (41.2% vs. 34%, p > 0.05).

Conclusions:

There is increased frequency of ACE and eNOS mutant allele carriage among Saudi patients affected with hypertension, particularly if accompanied by obesity and diabetes.

Keywords

ACE eNOS polymorphism hypertension Saudi Arabia

Introduction

It is clear – from family and epidemiological studies – that hypertension arises from a complex interplay between genetic and environmental lifestyle exposure and body weight.^1,2 Associated adiposity has been recognized to be controlled by several genes, with a heritability ranging from 25 to 40%.^3,4

The angiotensin-converting enzyme (ACE, EC 3.4.15.1) gene polymorphism is reported to be associated with hypertension, cardiovascular diseases, left ventricle hypertrophy, myocardial infarction and diabetes.^5,6 The ACE enzyme is a chloride and zinc-dependent dipeptidyl carboxypeptidase ubiquitously existing in mammalian species. As a bioactive component of the renin–angiotensin system (RAS) and kallikrein–kinin system (KKS), it plays a significant role in blood pressure regulation. It acts by hydrolyzing angiotensin I into a potent vasopressor peptide – angiotensin II – and also by deactivating the vasodepressor peptide bradykinin.⁷ The insertion/deletion (I/D) polymorphism of the ACE gene was characterized by the presence or absence of a 278-bp Alu repetitive sequence in intron 16. This polymorphism accounts for a large proportion of serum ACE activity, with the DD genotype having the highest and the II genotype having the lowest ACE activity.⁸

Endothelial nitric oxide synthase (eNOS) is one of three isoforms of nitric oxide synthase that exhibit homology of sequence and function.⁹ Nitric oxide (NO) is synthesized from L-arginine by a family of three enzymes, which are constitutive enzymes that produce low levels of basal NO production in a calcium-dependent fashion.^10,11 NO from the endothelium is considered an important atheroprotective mediator, as defects in generation of NO are associated with an increase in cardiovascular risk factors.¹² Multiple gene polymorphisms related to eNOS have been reported,¹³ but a common polymorphism exists in nucleotide 894 G-T that results in the conversion of glutamate to aspartate for codon 298 (E298D). The Asp298 or D variant has been shown to be associated with a shorter half-life of eNOS in endothelial cell culture, as the result of increased enzymatic cleavage.¹⁴ In addition, clinical studies have demonstrated that vascular responsiveness is altered in subjects with this variant, as patients with Asp298 have an increased vasoconstrictive response to phenylephrine consistent with decreased NOS activity;¹⁵ therefore, it has been implicated as a risk factor for both hypertension^14,16 and coronary disease.^17,18

Hypertension is a well-known prevalent disorder among the Saudi population, especially in Qassim Region which is a tribal region characteristically having high consanguinity rates plus relatively high aggregation rates for familial diseases such as obesity, hypertension and diabetes.^19,20 In spite of this fact, to date few data have been published concerning the genetic background of Saudi subjects in terms of their susceptibility to hypertension.

This study was planned in order to check for the association of ACE I/D and eNOS E298D genetic polymorphisms with hypertension among affected Saudi subjects.

Subjects and methods

As a pilot study ahead of a future wide-scale genetic analysis of Saudi patients with hypertension, a convenient sample of 120 affected subjects were included in this study. These were 80 males and 40 females with an age mean ±SD of 50.93 ± 15.43 years. They were known to have longstanding hypertension, i.e. with systolic blood pressure (SBP) between 140 and 200 mmHg, or diastolic blood pressure (DBP) between 90 and 120 mmHg. Their minimum duration of hypertension is 1 year, confirmed during at least two visits to the outpatient clinic of Buraydah Central Hospital, Qassim region, Saudi Arabia. Of these patients, 24.1% had a positive family history of hypertension, 32.4% had positive parental consanguinity, 12.8 % were smokers, 84.6% were obese, and 46.2% had diabetes. For comparison, 250 normal healthy unrelated subjects (142 males and 108 females) with an age mean and SD of 47.65 ± 11.15 years were taken from the same locality as controls.

Obesity was diagnosed on the basis of the most commonly used definitions, established by the World Health Organization (WHO) in 1997 and published in 2000, that defines the body mass index (BMI) of obesity as being 30 or greater.²¹ Type 2 diabetes was diagnosed on the basis of blood sugar during fasting (12–14 h) being higher than 126 mg/dl on at least two occasions, or with symptoms of hyperglycemia and a casual (random) plasma glucose higher than 200 mg/dl (11.1 mmol/l).²² Informed consent was obtained from all participants, as well as ethical approval from the local Ethics and Scientific Committees.

DNA extraction and amplification

The isolation of genomic DNA was done on a MagNA Pure LC instrument (Roche Molecular Biochemicals, Mannheim, Germany), using the manufacturer’s standard protocol. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of the corresponding polymorphism (TIB MolBiol; Berlin, Germany). The master mixture contained 2 μl of a 10× mixture of Light Cycler Fast-Start DNA master hybridization probes (Roche Diagnostics), 5 mM MgCl₂, 1 μM MTHFR primers including 0.075 μM of specific primers, and 0.2 μM of hybridization probes. Fluorescence curves were analyzed with the LightCycler software (version 3.5.3). Automated calculation of crossing points was done by the second-derivative maximum method. The fluorescence of each capillary was measured at wavelengths of 640 and 705 nm (dual-color option). Each run contained positive control, wild type (WT), mutant type (MT), heterozygous type (HT) and one negative control (blank reagent and water). Each result was confirmed by the specific peak in the corresponding melting curve.

Statistical analysis

Allelic and genotypic frequencies were compared and statistically analyzed using the Fisher’s exact test and odds ratio (OR) with 95% confidence intervals (95% CI). Conformity of genotype distributions with Hardy–Weinberg (HW) equilibrium was evaluated by chi-square analysis. For all tests, a p-value < 0.05 was considered to be statistically significant.

Results

Total cases of hypertension showed a significantly higher frequency of mutant D allele carriage (DD and DI) of the ACE gene (98.3% vs. 92.4%, p = 0.028), being approximately four times more prone to having hypertension (OR = 4.8, 95% CI = 1.1–21.2). In addition, cases showed significantly lower frequency of the wild type II genotype (1.7 vs. 7.6%). Interestingly, cases with hypertension, diabetes, and obesity showed 100% mutant D allele carriage that gave a just near-significant association (p = 0.052) (Table 1).

Table 1.

Frequency of ACE I/D genotypes among cases with hypertension, diabetes and obesity compared to controls.

Genotypes (ACE)	Controls	Total cases	Hypertension with obesity and diabetes
	n (%)	n (%)	n (%)
Total	250 (100)	120 (100)	51 (100)
Mutant (DD & ID)	231 (92.4)	118 (98.3)	51 (100)
Wild type (II)	19 (7.6)	2 (1.7)	0 (0)
p	–	0.028^*	0.052
OR (95% CI)	–	4.8 (1.1–21.2)	NA

NA: not applicable; *p significant <0.05

Regarding the eNOS E298D polymorphism (Table 2), the frequency of mutant eNOS D allele carriage was only observed to be higher than controls among cases with hypertension associated with diabetes and obesity, yet not reaching statistical significance (41.2% vs. 34%, p = 0.34). All other subgroups showed a non-significant difference of frequencies related to eNOS E298D gene polymorphism compared with controls.

Table 2.

Frequency of eNOS E298D genotypes among cases with hypertension, diabetes and obesity compared with controls.

Genotypes (eNOS)	Controls	Total cases	Hypertension with obesity and diabetes
	n (%)	n (%)	n (%)
Total	250 (100)	120 (100)	51 (100)
Mutant (DD & DE)	85 (34)	39 (32.5)	21 (41.2)
Wild type (EE)	165 (66)	81 (67.5)	30 (58.8)
p	–	0.81	0.34
OR (95% CI)	–	0.9 (0.59–1.5)	1.4 (0.7–2.6)

Interestingly, testing for the association of hypertension with combined genotypes of the two polymorphisms revealed only a significant association with ACE D allele carriage (data not shown). Comparing case subgroups in terms of risk factors such as gender, age, smoking, consanguinity and family history showed a non-significant difference related to all studied genotypes and alleles (Table 3).

Table 3.

Genetic polymorphisms of ACE I/D and eNOS E298D genes related to potential risk factors of hypertension.

	ACE I/D			eNOS E298D
	DD+ID	II	p	DD+DE	EE	p
Sex (%)
Male (n=80)	98.7	1.3	>0.05	69.7	30.3	>0.05
Female (n=20)	97.4	2.6		62.2	37.8
Age (years, mean ± SD)
	56.9 ± 18.5	50.0 ± 21.05	>0.05	58.4 ± 20.5	54.8 ± 17	>0.05
Smoking (%)
Positive (n=14)	100	0.0	>0.05	90.9	9.1	>0.05
Negative (n=106)	98.9	1.1		64.3	35.7
Family history (%)
Positive (n=29)	100	0.0	>0.05	56.5	43.5	>0.05
Negative (n=91)	98.9	1.2		70.1	29.9
Consanguinity (%)
Positive (n=37)	97.1	2.9	>0.05	6.25	37.5	>0.05
Negative (n=83)	100	0.0		68.7	31.3

Discussion

This study showed that Saudi patients with hypertension, particularly when associated with obesity and diabetes, had significantly higher frequency of the mutant ACE D allele carriage rate than controls, with a significantly higher frequency of the heterozygous polymorphism ID. Interestingly, the ACE DD genotype frequency in Qassim region (51.7%) was higher than reported in other Mediterranean and Asian countries such as Egypt (48.9%),²³ Turkey (27.5%),²⁴ France (30.6%),²⁵ Japan (29.03%)²⁶ and Korea (25%).²⁷ On the other hand, hypertensive cases associated with obesity and cases with hypertension alone showed higher frequencies of the mutant D allele carriage than controls, but this was statistically non-significant (p > 0.05).

These findings are in agreement with those of Kim, who noted that there was a trend towards association of the ACE I/D polymorphism with hypertension but not with obesity among Korean women.²⁸ Also, Park et al. found that the D allele is associated with an increased level of ACE in Korean hypertensive adolescents.²⁹ Niu et al. reported that the interactive haplotypes of genes determining the RAS, including the ACE genes, were associated with hypertension among the Fangshan Chinese population.³⁰ Similar studies among different populations have provided strong evidence for the association of the ACE I/D genotype in hypertensive cases with type 2 diabetic subjects without obesity, such as those done in Tunsian,³¹ Malaysian³² and Chinese³³ subjects. Conversely, many studies have pointed out that DD genotypes failed to show an association with hypertension, such as those done among Chinese,³³ Punjabi Pakistanis,³⁴ a rural Indian population of Haryana³⁵ and in the western Black Sea region of Turkey.³⁶ Similarly, no association was found regarding the ACE I/D polymorphism among cases with hypertension and diabetes as reported among Iranians,³⁷ French,³⁸ Japanese³⁹ and Slovak populations.⁴⁰

It has been speculated that ACE and the components of the RAS are expressed in adipose tissue and therefore might be associated with obesity. Wacker et al. indicated that the ACE I/D polymorphism may be differentially associated with obesity depending on multiple factors.⁴¹ However, Cooper et al. pointed out that the I/D polymorphism of the ACE gene was associated with variation in the levels of ACE, but inconsistently with BMI. Therefore, the investigators suggested that obesity may alter the levels of ACE and angiotensinogen, and provide a potential pathway through which obesity leads to elevation of blood pressure.⁴² In this respect, Kramer et al. examined the association between obesity and 13 ACE gene polymorphisms, including the I/D polymorphism, and found no polymorphism consistently over-transmitted from parents to obese offspring among blacks living in Ibadan, Nigeria; Spanish Town, Jamaica; and Chicago.⁴³

Regarding the eNOS E298D polymorphism, the frequency of mutant D allele carriage was only observed to be higher in cases of hypertension associated with diabetes and obesity as compared with controls, yet was statistically non-significant. These results do not support the hypothesis that the E298D eNOS polymorphism contributes to the genetic susceptibility to hypertension with or without obesity and diabetes among Saudi subjects. Similarly, many studies have pointed out that the E298D eNOS polymorphism failed to show an association in hypertension among different populations, for instance, in a population-based cohort of Caucasians,⁴⁴ in Anglo-Celtic whites,⁴⁵ in Japanese⁴⁶ and in Pakistani populations.⁴⁷ In addition, a meta-analysis by Zintzaras et al. has confirmed no association of the E298D eNOS polymorphism with either hypertension status or blood pressure levels in other populations.⁴⁸ Conversely, there are studies showing a positive association of E298D eNOS with hypertension in Caucasians,¹⁶ Japanese,⁴⁹ Koreans,⁵⁰ Chinese⁵¹ and in male Asian Indians.⁵² Such controversial associations might lead us to believe that final conclusions are limited by diverse factors challenging human geneticists involved in multifactorial disease research. It is relevant to recognize that disease prevalence is a consequence of the intersection of the genetic variation that represents a population with different environmental exposures. Each population is expected to have a different distribution of relative genotype frequencies and a different constellation of possible environmental histories.⁵³

In this respect, to clarify the significance of this gene polymorphism, we recommend a large-scale prospective cohort study to more accurately judge associations of the polymorphism with hypertension. Future research should preferentially focus on gene–environment interactions as well as haplotype analyses.

In conclusion, this study has demonstrated the possible association of ACE I/D (but not the eNOS E298D) gene polymorphism with hypertension in a Saudi population from Qassim Region. This polymorphism can be used as marker for the susceptibility to hypertension among affected families, who can be counseled to avoid other interactive environmental risk factors.

Footnotes

Acknowledgements

Authors are grateful for the Research Deanship, Qassim University, Saudi Arabia for funding this work.

Conflict of interest

None declared.

Funding

This research was funded by the Research Deanship, Qassim University, Saudi Arabia.

References

Snieder

Harshfield

Treiber

. Heritability of blood pressure and hemodynamics in African- and European-American youth. Hypertension 2003; 41: 1196–1201.

Daniels

Kardia

Hanis

. Genetic Epidemiology Network of Arteriopathy study. Familial aggregation of hypertension treatment and control in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. Am J Med 2004; 116: 676–681.

Hagberg

Rankinen

Loos

. Advances in exercise, fitness and performance genomics in 2010. Med Sci Sports Exerc 2011; 43: 743–752.

Rankinen

Zuberi

Chagnon

. The human obesity gene map: the 2005 update. Obesity (Silver Spring) 2006; 14: 529–644.

Morshed

Khan

Akhteruzzaman

. Association between angiotensin I-converting enzyme gene polymorphism and hypertension in selected individuals of the Bangladeshi population. J Biochem Mol Biol 2002; 35: 251–254.

Kennon

Petrie

Small

. Angiotensin converting enzyme gene and diabetes mellitus. Diabet Med 1999; 16: 448–458.

Zhao

. Structure and function of angiotensin converting enzyme and its inhibitors. Sheng Wu Gong Cheng Xue Bao 2008; 24: 171–176.

Rigat

Hubert

Alhenc-Gelas

. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86: 1343–1346.

Forstermann

Nakane

Tracey

. Isoforms of nitric oxide synthase: functions in the cardiovascular system. Eur Heart J 1993; 14(Suppl I): 10–15.

10.

Stuehr

. Structure-function aspects in the nitric oxide synthases. Annu Rev Pharmacol Toxicol 1997; 37: 339–359.

11.

Moncada

Higgs

. The L-arginine-nitric oxide pathway. N Engl J Med 1993; 329: 2002–2012.

12.

Vallance

Collier

Moncada

. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989; 2: 997–1000.

13.

Marsden

Heng

Scherer

. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993; 268: 17478–17488.

14.

Jáchymová

Horký

Bultas

. Association of the Glu298Asp polymorphism in the endothelial nitric oxide synthase gene with essential hypertension resistant to conventional therapy. Biochem Biophys Res Commun 2001; 284: 426–430.

15.

Shimasaki

Yasue

Yoshimura

. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. J Am Coll Cardiol 1998; 31: 1506–1510.

16.

Lacolley

Gautier

Poirier

. Nitric oxide synthase gene polymorphism, blood pressure and aortic stiffness in normotensive and hypertensive subjects. J Hypertens 1998; 16: 31–35.

17.

Granath

Taylor

van Bockxmeer

. Lack of evidence for association between endothelial nitric oxide synthase gene polymorphisms and coronary artery disease in the Australian Caucasian population. J Cardiovasc Risk 2001; 8: 235–241.

18.

Miyamoto

Saito

Kajiyama

. Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension 1998; 32: 3–8.

19.

Soyannwo

Kurashi

Gadallah

. Blood pressure pattern in Saudi population of Gassim. Afr J Med Med Sci 1998; 27: 107–116.

20.

Al-Sheikh

Aljabri

Al-Ansary

. Prevalence of and risk factors for peripheral arterial disease in Saudi Arabia. A pilot cross-sectional study. Saudi Med J 2007; 28: 412–414.

21.

WHO. http://www.who.int/mediacentre/factsheets/fs311/en/index.html (2000, accessed September 2011)

22.

American Diabetes Association. Standards of medical care in diabetes – 2009. Diabetes Care 2009; 32(Suppl 1): S13–S61.

23.

Ulu

Elsobky

Elsayed

. Frequency of five thrombophilic polymorphisms in the Egyptian population. Turk J Hematol 2006; 23: 100–103.

24.

Eroglu

Cetinkalp

Erdogan

. Association of the angiotensinogen M235T and angiotensin-converting enzyme insertion/deletion gene polymorphisms in Turkish type 2 diabetic patients with and without nephropathy. J Diabetes Complications 2008; 22: 186–190.

25.

Marre

Bernadet

Gallois

. Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes 1994; 43: 384–388.

26.

Mizuiri

Hemmi

Inoue

. Angiotensin-converting enzyme polymorphism and development of diabetic nephropathy in non-insulin-dependent diabetes mellitus. Nephron 1995; 70: 455–459.

27.

Shin

Park

. Relationships between angiotensin I converting enzyme gene polymorphism and renal complications in Korean IDDM patients. Korean J Intern Med 1996; 11: 133–137.

28.

Kim

. Association of angiotensin-converting enzyme insertion/deletion polymorphism with obesity, cardiovascular risk factors and exercise-mediated changes in Korean women. Eur J Appl Physiol 2009; 105: 879–887.

29.

Park

Ahn

Lee

. Insertion/deletion polymorphism of angiotensin converting enzyme gene in Korean hypertensive adolescents. Heart Vessels 2009; 24: 193–198.

30.

Niu

Hou

. Haplotype-based association of the renin-angiotensin-aldosterone system genes polymorphisms with essential hypertension among Han Chinese: the Fangshan study. J Hypertens 2009; 27: 1384–1391.

31.

Mehri

Koubaa

Hammami

. Genotypic interactions of renin-angiotensin system genes with diabetes type 2 in a Tunisian population. Mehri Life Sci 2010; 87: 49–54.

32.

Ramachandran

Ismail

Stanslas

. Association of insertion/ deletion polymorphism of angiotensin-converting enzyme gene with essential hypertension and type 2 diabetes mellitus in Malaysian subjects. J Renin Angiotensin Aldosterone Syst 2008; 9: 208−214.

33.

Chiang

Lai

Chern

. Lack of association of the angiotensin converting enzyme gene polymorphism with essential hypertension in a Chinese population. Am J Hypertens 1997; 10: 197–201.

34.

Alvi

Hasnain

. ACE I/D and G2350A polymorphisms in Pakistani hypertensive population of Punjab. Clin Exp Hypertens 2009; 31: 471–480.

35.

Gupta

Agrawal

Goel

. Angiotensin-converting enzyme gene polymorphism in hypertensive rural population of Haryana. India J Emerg Trauma Shock 2009; 2: 150–154.

36.

Tascilar

Dursun

Ankarali

. Angiotensin-converting enzyme insertion/deletion polymorphism has no effect on the risk of atherosclerotic stroke or hypertension. J Neurol Sci 2009; 285: 137–141.

37.

Nikzamir

Nakhjavani

Golmohamadi

. Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with metabolic syndrome in Iranians with type 2 diabetes mellitus. Arch Iran Med 2008; 11: 3–9.

38.

Hadjadj

Fumeron

Roussel

. Prognostic value of the insertion/deletion polymorphism of the ACE gene in type 2 diabetic subjects. Diabetes Care 2008; 31: 1847–1852.

39.

Daimon

Oizumi

Saitoh

. The D allele of the angiotensin-converting enzyme insertion/deletion (I/D) polymorphism is a risk factor for type 2 diabetes in a population based Japanese sample. Endocr J 2003; 50: 393–398.

40.

Danková

Siváková

Luptáková

. Association of ACE (I/D) polymorphism with metabolic syndrome and hypertension in two ethnic groups in Slovakia. Anthropol Anz 2009; 67: 305–316.

41.

Wacker

Godard

McCabe

. Sex difference in the association of the angiotensin converting enzyme I/D polymorphism and body mass index. Med Sci Monit 2008; 14: CR353–CR357.

42.

Cooper

McFarlane-Anderson

Bennett

. Angiotensinogen and obesity: a potential pathway leading to hypertension. J Hum Hypertens 1997; 11: 107–111.

43.

Kramer

Kan

. Angiotensin-converting enzyme gene polymorphisms and obesity: an examination of three black populations. Obes Res 2005; 13: 823–828.

44.

Wolff

Grabe

Schlüter

. Endothelial nitric oxide synthase Glu298Asp gene polymorphism, blood pressure and hypertension in a general population sample. J Hypertens 2005; 23: 1361–1366.

45.

Benjafield

Morris

. Association analyses of endothelial nitric oxide synthase gene polymorphisms in essential. Am J Hypertens 2000; 13: 994–998.

46.

Kishimoto

Misawa

Kaetu

. eNOS Glu298Asp polymorphism and hypertension in a cohort study in Japanese. Prev Med 2004; 39: 927–931.

47.

Khawaja

Taj

Ahmad

. Association of endothelial nitric oxide synthase gene G894T polymorphism with essential hypertension in an adult Pakistani Pathan population. Int J Cardiol 2007; 116: 113–115.

48.

Zintzaras

Kitsios

Stefanidis

. Endothelial NO synthase gene polymorphisms and hypertension: a meta-analysis. Hypertension 2006; 48: 700–710.

49.

Tsujita

Baba

Yamauchi

. Association analyses between genetic polymorphism of endothelial nitric oxide synthase gene and hypertension in Japanese. The Suita study. J Hypertens 2001; 19: 1941–1948.

50.

Lustberg

Schwartz

Lee

. The G894–T894 polymorphism in the gene for endothelial nitric oxide synthase and blood pressure in lead-exposed workers from Korea. J Occup Environ Med 2001; 16: 581–590.

51.

Tang

Yang

Wang

. Association between a G894T polymorphism of eNOS gene and essential hypertension in Hani and Yi minority groups of China. Arch Med Res 2008; 39: 222–225.

52.

Srivastava

Narang

Sreenivas

. Association of eNOS Glu298Asp gene polymorphism with essential hypertension in Asian Indians. Clin Chim Acta 2008; 387: 80–83.

53.

Sing

Stengard

Kardia

. Genes, environment, and cardiovascular disease. Arterioscler Thromb Vasc Biol 2003; 23: 1190–1196.