Genetic determinants of 5-lipoxygenase pathway in a Spanish population and their relationship with cardiovascular risk

Atherosclerosis 224 (2012) 129e135

Contents lists available at SciVerse ScienceDirect

Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Genetic determinants of 5-lipoxygenase pathway in a Spanish population and their relationship with cardiovascular risk Mercedes Camacho a, Angel Martinez-Perez b, Alfonso Buil b, Laura Siguero a, Sonia Alcolea a, Sonia López b, Jordi Fontcuberta c, Juan-Carlos Souto c, Luis Vila a, *,1, José-Manuel Soria b, **,1 a b c

Laboratory of Angiology, Vascular Biology and Inflammation, Institute of Biomedical Research (II-B Sant Pau), Barcelona, Spain Unit of Genomic of Complex Diseases, Institute of Biomedical Research (II-B Sant Pau), Barcelona, Spain Department of Hematology, Institute of Biomedical Research (II-B Sant Pau), Barcelona, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 July 2011 Received in revised form 25 June 2012 Accepted 1 July 2012 Available online 13 July 2012

Objective: Leukotrienes (LT) play a role in inflammation, cardiovascular diseases, and cancer. Although some studies suggest that there are genes that determine variability of some LT-related phenotypes, the genetic influence on these phenotypes has not been evaluated. Methods: The relative contributions of genetic and environmental influences to the 5-lipoxygenase pathway-related phenotypes (5-Lipoxygenase, five lipoxygenase activating protein (FLAP), LTA4-hydrolase and LTC4-synthase expression, and LTB4-plasma concentration and LTB4 production by stimulated whole blood) were assessed in a sample of 934 individuals in 35 extended families. Our design is based on extended families recruited through a probands with idiopathic thrombophilia. This strategy allows us the analysis of the effects of measured covariates (such as sex, age and smoking), genes, and environmental variables shared by members of a household. Results: All of these phenotypes showed significant genetic contributions, with heritabilities ranging from 0.33 to 0.51 for enzyme expression and from 0.25 to 0.50 for LTB4 production of the residual phenotypic variance. Significant phenotypic and genetic correlation among the LT-related traits was found. More importantly, FLAP and LTA4-hydrolase expression exhibit significant genetic correlations with arterial thrombosis, indicating that some of the genes that influence quantitative variation in these phenotypes also influence the risk of thrombosis. Conclusion: This is the first study that quantifies the genetic component of 5-Lipoxygenase pathway phenotypes. The high heritability of these traits and the significant genetic correlations between arterial thrombosis and some of these phenotypes suggest that the exploitation of correlated quantitative phenotypes will aid the search for susceptibility genes. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Thrombosis 5-lipoxygenase LTA4-hydrolase LTC4-synthase FLAP Leukotrienes

1. Introduction Leukotrienes (LTs) are important inflammatory mediators and targets for pharmacological intervention. The biosynthesis of LTs in the vasculature depends strongly on leukocyte recruitment and

Abbreviations: COX, cyclooxygenase; EIA, specific enzyme immunoassay; FLAP, five lipoxygenase activating protein; 5-LO, 5-lipoxygenase; LTA4-H, LTA4-hydrolase; LTC4-S, LTC4-synthase. * Corresponding author. H. S. Creu i S. Pau, S. Antonio Ma Claret 167, 08025 Barcelona, Spain. Tel.: þ34 93 5565705; fax: þ34 93 4552331. ** Corresponding author. Unitat de Genomica de Malalties Complexes, Institut de Recerca, H. S. Creu i S. Pau S. Antonio Ma Claret 167, 08025 Barcelona, Spain. Tel.: þ34 93 5537656; fax: þ34 93 5565524. E-mail addresses: [email protected] (L. Vila), [email protected] (J.-M. Soria). 1 These authors contributed equally to this work. 0021-9150/$ e see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2012.07.001

activation, and on cellecell interaction between leukocytes and vascular cells in the inflamed areas. The common precursor of LTs, LTA4, is formed from arachidonic acid (AAc) by the 5-lipoxygenase (5-LO). The 5-LO activity depends on ATP and Ca2þ, the latter promotes binding of 5-LO to a nuclear membrane associated protein so-called Five Lipoxygenase Activating Protein (FLAP). LTA4 is subsequently conversed into either LTB4 or LTC4, by the action of LTA4-hydrolase (LTA4-H) and LTC4-synthase (LTC4-S), respectively. A scheme of Leukotrienes biosynthesis pathway is shown in Fig. 1(for reviews see [1,2]). Activated leukocytes, the restricted cell lines that express 5-LO and FLAP, are a predisposing factor to atherosclerosis, unstable angina and cardiac infarction. The most important biological actions of peptide-LTs are vaso-constriction and bronchoconstriction and increase in vascular permeability. The LTB4 is one of the most powerful endogenously synthesized chemotactic

130

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135

Membrane phospholids Phospholipases (mainly A2) COOH

AA 5-LO/FLAP

OH

OOH

COOH

COOH

5-HPETE 5-HETE

5-LO/FLAP O

HO

LTC4-S

LTA4

COOH

N

NH

COOH

OH

COOH

O

LTA4-H

COOH

LTB4

NH O

S OH

COOH

LTC4 COOH

O

HOOC

NH

NH

NH

S

2. Material and methods

S OH

LTE4

COOH

atherothrombotic cerebral infarction in a Japanese population with metabolic syndrome [18]. In addition, one SNP in the LTC4-S gene was related to coronary artery calcium and carotid intima media thickness in young women [19]. However, other studies have reported that this SNP has a decreased risk for ischemic cerebrovascular events in different populations [20,21]. Moreover, another SNP 1072 bp upstream in the LTC4-S promoter has been reported to confer an increased risk of ischemic cerebrovascular disease [21]. Comparatively, none of the studies have attempted to quantify the nature and extent of genetic determinants of phenotypic variation in LTs-related traits through the use of family-based sampling designs. In this context, there is increasing evidence in favor of the implication of 5-LO pathway in cardiovascular pathology, but little is known about the underlying factors involved in the determination of LT-biosynthetic pathway. The primary purpose of the present investigation was to examine the relative roles of genetic and environmental factors in determining LT-related phenotypes. To this end we studied a sample of extended Spanish families ascertained through individuals with thrombophilia. To our knowledge, this study is the first large-scale family study of the genetics of quantitative variation in these risk factors for inflammatory pathological conditions.

OH

COOH

2.1. Enrollment of family members and blood collection

LTD4

Peptide-LTs Fig. 1. Schematic representation of 5-lipoxygenase pathway. Formation of leukotrienes from arachidonic acid (AAc) is depicted. LT, leucotriene; FLAP, five lipoxygenase activating protein; LTA4-H, LTA4-hydrolase; LTC4-S, LTC4-synthase.

factors for polymorphonuclear neutrophils (PMN) [3] and also activates and increases their survival at the inflammation site [4]. It has also been reported that expression of LTB4 biosynthetic machinery confers plaque instability [5]. The involvement of LTB4 and peptide-LTs, and PMN in cardiovascular physiopathology has been demonstrated in animal models and patients [1,2,6e10]. More importantly, association studies of the genes that encode 5-LO pathway-related enzymes have provided some evidence that this pathway plays a role in cardiovascular disease. Several polymorphisms identified in a GC-rich sequence of the ALOX5 gene promoter (encode 5-LO) [11], have been associated with atherosclerosis [12]. Additionally, a single nucleotide polymorphism (SNP) in the ALOX5 gene promoter was associated with coronary artery disease [13]. In contrast, other studies reported no association of this tandem Sp1/Egr1 binding repeat with myocardial infarction [14]. On the other hand, Helgadottir et al. [15] identified a four-SNP haplotypes in the FLAP gene associated with a greater risk of myocardial infarction and stroke. They reported also that male carriers of one of these haplotypes with a history of myocardial infarction have an increased LTB4 production from ex vivo-stimulated neutrophils. Nevertheless, subsequent studies gave controversial results (reviewed in Ref. [16]). In addition, it has been reported that a haplotype of five SNPs in the LTA4-H gene (more frequent in subjects with a history of cardiovascular disease) confers ethnicity-specific risk of myocardial infarction [17]. Furthermore, a positive correlation was observed between this haplotype and LTB4-formation in ex vivo-stimulated granulocytes [17]. Subsequent studies reported that two of the SNPs of this LTA4-H haplotype were associated independently with

The Spanish families available for our studies were a new set of extended families from the GAIT (genetic analysis of idiopathic thrombophilia) project [22]. This new set of families included 934 individuals belonging to 35 extended families. The criteria for inclusion in the study have been described previously [22]. To be included, a family had to have at least 10 living individuals in three or more generations. Families were selected through a proband with idiopathic thrombophilia, which was defined as multiple thrombotic events (at least one spontaneous), a single spontaneous episode of thrombosis with a first-degree relative also affected, or onset of thrombosis before age 45. The proband’s thrombophilia was considered idiopathic because all known biological causes (e.g., antithrombin deficiency, Protein S and C deficiencies, activated protein C resistance, plasminogen deficiency, heparin cofactor II deficiency, Factor V Leiden, dysfibrogenemia, lupus anticoagulant, and antiphospholipid antibodies) of thrombophilia were excluded. The subjects were interviewed by a physician to determine their health and reproductive history, current medications, alcohol consumption, physical activity, and use of sex hormones (oral contraceptives or hormonal replacement therapy). Also, they were questioned about previous episodes of venous or arterial thrombosis and the age at which these events occurred, as well as potentially correlated disorders such as diabetes, lipid disease, asthma, allergic rhinitis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and cancer. All protocols were reviewed by the Institutional Review Board of the Hospital de Sant Pau (Barcelona). Adult subjects gave informed consent for themselves and for their minor children. The depth and complexity of the pedigrees are illustrated by the number of pairs of relatives contained therein (Table 1). Among the 934 individuals, 7 people were excluded because they took steroids, 2 because took non-steroid anti-inflammatory drugs within the 15 days previous to the blood extraction, 3 because both of the previous causes and 2 because they took chemotherapy drugs. Within the remaining 920 individuals, 8 were excluded for the LTB4-plasma and LTB4-A23187 because LTB4 levels were under the limit of detection, other 19 individuals were excluded from 5-LO, FLAP, LTA4-H and LTC4-H because the poor

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135 Table 1 Description of the family member relationship based on the number of relative pairs. n

Relation

Degree of relation

1162 1528 693 1296 1488 766 709 1357 1700 159 1075 212

Self Parent-offspring Siblings Grandparent-grandchild Avuncular Great grandparent-grandchild Grand avuncular 1st cousins 1st cousins, 1 removed 1st cousins, 2 removed 2nd cousins 2nd cousins, 2 removed

0 1 1 2 2 3 3 3 4 5 5 6

131

Laboratories, Inc., Houston, TX, USA) according to the manufacturer’s instructions. Then cDNA was prepared by reverse transcribing 1 mg RNA per 20 mL with High-Capacity cDNA Archive kit with random hexamers (Applied Biosystems, Foster City, CA). The mRNA expression of 5-LO, FLAP, LTA4-H and LTC4-S genes was determined by Real-Time-PCR in an ABI Prism 7000 using predesigned validated assays (TaqMan Gene Expression Assays; Applied Biosystems) and universal thermal cycling parameters. Relative mRNA expression was expressed as transcript/b-actin ratios. 2.3. Whole blood assays

maintenance of RNA, and finally one more was excluded in the 5-LO because it was and outlier in the distribution (Table 2). A sample of 10 mL of peripheral venous blood was collected from all of the participants in heparin-containing tubes and aliquoted for mRNA analysis and for the whole blood assays. Total mRNA extraction and storage were performed according to standard protocols. EDTA and citrate samples were obtained for whole blood cell counts. Whole blood cell counts were performed in the standard hematology analyzer Sysmex XE-2100Ò (Roche Diagnostics). The results included: total leukocyte count, absolute monocyte count, platelet count, and plateletcrit or total platelet volume. In addition, we obtain the leukocyte formula also.

An aliquot of 1.5 mL of heparinized whole blood was warmed at 37  C prior to the addition of an ethanolic solution of calcium ionophore A23187 (Sigma) to yield a final concentration of 25 mmol/ L. Blood was then incubated at 37  C for 10 min. Afterward, the blood was immediately centrifuged at 4  C and the supernatant was stored at 80  C until analyzed. LTB4 production was estimated by the difference of LTB4 levels after and before stimulation with A23187. Before stimulation LTB4 was measured in the plasma that was obtained as described above. 2.4. Analysis of LTB4 LTB4 concentration was analyzed by specific enzyme immunoassays (EIA, Caymam Chemical, Ann Arbor, MI) following the manufacturer’s instructions. 2.5. Statistical methods

2.2. 5-LO, FLAP, LTA4-H and LTC4-S mRNA analysis 3.5 mL of anticoagulated peripheral venous blood was centrifuged at 1300  g for 10 min at room temperature. The pellet was washed with PBS buffer at pH 7.4. Erythrocytes were lysed using 50 mL of TriseHCl 20 mM, 5 mM MgCl2.6H2O pH 7.5 to the pellet and incubating for 10 min in an ice-water bath. After centrifugation at 1600  g for 15 min, the pellet was washed with TriseHCl. The pellet was collected in 1 mL of Ultraspec (Biotecx Laboratories, Inc, Houston, Texas, USA) and stored at 80  C. For mRNA analysis, total RNA was extracted from the cell pellet by chloroform/isopropanol precipitation using Ultraspec (Biotecx

The statistical methods used in our study have been described previously [23]. Briefly, the phenotypic covariance among relatives was used to estimate the additive genetic and shared environmental components of variance. Fixed effects included female sex, age, smoking and depending on traits, use of non-steroid antiinflammatory drugs and number of leukocytes and platelets. Discrete covariate (female sex) was scaled so that the regression coefficients represented the effect of the covariate versus its absence. Maximum likelihood methods were used to estimate simultaneously the means and variances as well as the effects of covariates,

Table 2 Description of the sample with the quantitative value of phenotypes. Results are the mean  SD of values of phenotypes obtained from all subjects (all), males and females. Phenotype

n

Units

All

Male

Female

Age Smoking Sex Venous thrombosisa Arterial thrombosisb Venous or arterial thrombosis 5-LOc 5-LOc/106 PMN FLAPc FLAPc/106 PMN LTA4-Hc LTC4-Sc LTB4-plasma LTB4-A23187 LTB4-A23187/106 PMN LTB4-A23187/106 MØd

920 920 920 920 920 920 900 900 901 901 901 901 912 912 912 912

Years Number Number Number Number Number e e e e e e pg/mL ng/mL ng ng

39.32  21.35 224 (24.35%)

37.74  21.39 108 (23.68%) 456 (49.6%) 29 (6.36%) 24 (5.26%) 49 (10.75%) 114.41  69.93 33.53  21.13 20.43  15.35 5.86  4.28 28.64  18.27 0.34  0.18 90.37  71.28 113.99  109.91 30.86  25.60 222.8  197.6

40.87  21.21 116 (25.00%) 464 (50.4%) 52 (11.21%) 21 (4.53%) 64 (13.79%) 117.60  71.10 31.97  18.02 19.83  14.53 5.37  3.45 25.39  15.87 0.33  0.17 87.53  69.63 84.07  81.22{ 22.55  20.55{ 191.1  190.5x

81 (8.80%) 45 (4.89%) 113 (12.28%) 116.29  69.38 32.74  19.48 20.03  14.38 5.65  3.87 27.20  16.99 0.34  0.18 89.25  70.39 97.65  96.16 26.30  23.33 205.1  192.1

xp < 0.01; {p < 0.001 when compared with males. a Venous thrombosis defined as deep venous thrombosis, pulmonary embolism, superficial thrombophlebitis or other venous thromboses events. b Arterial thrombosis defined as myocardial infarction, angina pectoris, ischemic stroke, transient ischemic attack, peripheral artery or other arterial thromboses events. c b-actin relative expression; 2DCt  1000. d MØ, monocytes.

132

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135

heredity, and household effect, using the computer package SOLAR [24]. The significance of covariate effects as well as the significance of genetic and household effects was assessed by means of the likelihood-ratio test. Estimates of variance component parameters, including the heritabilities of the quantitative measures and all the phenotypic, genetic, and environmental correlations between venous and arterial thrombosis and the quantitative phenotypes, were obtained by use of maximum-likelihood estimation. All hypothesis tests were performed using likelihood-ratio test statistics [25] implemented in the SOLAR computer package. This analysis allowed the partitioning of the phenotypic correlations among these traits into factors due to common genetic influences and common environmental influences [24]. Because the pedigrees were ascertained through a thrombophilic proband, all analyses included an ascertainment correction to allow unbiased estimation of parameters relevant to the general population. To achieve this, the likelihood for each family ascertained through a thrombophilic proband was conditioned on the phenotype of the proband.

3.2. Contribution of genetics to the variability in the 5-LO pathway phenotypes The components of variance are shown in Table 3. They are based upon the most parsimonious model (i.e., the model that exhibits the minimum complexity) for each phenotype, including significant sources of variation only. The remaining variance not accounted for in Table 3 is attributable to individual-specific random environmental influences and random error. The levels of expression of the enzymes showed highly significant heritabilities, ranging from 0.325 to 0.510 after correcting for covariate effects. The proportion of the residual phenotypic variability accounted for shared household effects tended to be considerably smaller than that accounted for by the genetic effects. This indicates that genes are important in determining the expression of these traits. Heritability of basal levels of LTB4 was highly significant, whereas LTB4 production in response to A23187 have a moderate heritability either when expressed in absolute values or when normalized for the number of PMN or monocytes (Table 3). 3.3. Phenotypic and genetic correlation among leukotriene-related traits

3. Results 3.1. Characteristics of the population and samples Table 2 shows the characteristics of the sample and the values of the phenotypes that were analyzed. The ages ranged from 3 to 101 years and the number of male and female subjects was similar with a mean age and range. Since LTs are initially produced by leukocytes, we analyzed first the statistical correlation among all of the enzyme phenotypes and LTB4 levels with the total number of leukocytes, PMN and eosinophils. Statistically significant positive correlation was found between 5-LO, FLAP expression, and A23187stimulated production of LTB4 with PMN number (R ¼ 0.27, p ¼ 2.31$1019; R ¼ 0.23, p ¼ 6.63$1012 and R ¼ 0.23, p ¼ 9.08$1017 respectively). No correlation of any other parameter with PMN number was found. Since monocytes also express 5-LO, correlations of mRNA parameters, plasma levels of LTB4 or A23187stimulated production of LTB4 with monocyte number were examined also. We observed only the correlation between monocyte number and A23187-stimulated production of LTB4 (R2 ¼ 0.198, p ¼ 4.6$109). The number of monocyte did no correlate with any other parameter. Therefore, data concerning 5-LO and FLAP expression, and A23187-stimulated production of LTB4 were not only expressed as total value but also as a value normalized by the PMN density, and A23187-stimulated production of LTB4 was also expressed normalized for the monocyte density. Significant differences were observed between males and females in the production of LTB4 in both, A23187-stimulated and normalized by the PMN or monocyte density (Table 2).

The expression value of all of the enzymes (5-LO, FLAP, LTC4-S and LTA4-H) showed significant positive phenotypic correlation among all of them (Table 4). The largest phenotypic correlations (jrpj > 0.5) were seen between 5-LO with FLAP and LTC4-S. When phenotypic correlations were partitioned using a bivariant variance component model in terms of genetic and environmental correlations, all of the parameters exhibited environmental correlations (Table 4) but only 5-LO, FLAP and LTC4-S exhibited significant genetic correlations. In other words, the phenotypic quantitative variation of LTA4-H is mainly due to environmental effects. However, the correlations between the expression of enzymes involved in the biosynthesis of LTB4 and the LTB4 production (basal plasma levels or calcium ionophore-induced levels) showed no phenotypic or genetic significance. 3.4. Association between the phenotypes and the disease status Table 4 shows the results of bivariate genetic analyses of disease status (venous or arterial thrombosis), with each of the leukotriene pathway-related parameters considered. Of these, we found a significant phenotypic, genetic and environmental correlation between FLAP expression and arterial thrombosis (Table 4). It is interesting to note that we found no statistically significant correlation for venous thrombosis. In addition, although the LTA4-H expression exhibits also significant phenotypic correlation with arterial thrombosis, the coefficient of this correlation (0.008) is

Table 3 Components of variance from the most parsimonious model. Results are the mean  SE of values of phenotypes. The last column is the proportion of variance due to all final covariates. Phenotype

n

Heritability (h2)

5-LO 5-LO/106 PMN FLAP FLAP/106 PMN LTA4-H LTC4-S LTB4-plasma LTB4-ion LTB4-ion/106 PMN LTB4-ion/106 MØa

900 900 901 901 901 901 912 912 912 912

0.510 0.362 0.385 0.325 0.452 0.369 0.503 0.302 0.319 0.247

a

MØ, monocytes.

         

0.065 0.061 0.068 0.063 0.067 0.069 0.057 0.077 0.061 0.076

p-values (h2)

Household (c2)

7.62$1016 1.68$1012 1.24$1011 6.09$1010 3.09$1013 1.28$1009 6.14$1027 2.80$1006 3.15$1010 7.8$1005

0.102 0 0.207 0 0.179 0.110 0 0.218 0 0.245

 0.044  0.049  0.048  0.047  0.053  0.056

p-values (c2)

Covariates

% Variance due to covariates

6.55$1003 1.42$1002 1.70$1006 1.11$1005 1.68$1005 5.37$1003

Age, smoking Age, sex, smoking Age, smoking Age, sex, smoking Age, sex Age, smoking Smoking Age, sex, smoking Age, sex, smoking Age, sex, smoking

1.40 7.32 3.88 9.37 0.52 0.76 0.62 5.09 8.61 2.67

1.70$1006 1.43$1003 4.0$1007

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135

133

Table 4 Regression coefficient (p-value) of phenotypic correlations between the expressions of the enzymes and thrombosis. The phenotypical correlations were partitioned in terms of genetic (G) and environmental (E) correlations. Phenotype

LTC4-S

5-LO 5-LO-(G) 5-LO-(E) FLAP FLAP-(G) FLAP-(E) LTA4-H LTA4-H-(G) LTA4-H-(E)

0.549 0.483 0.632 0.434 0.441 0.432 0.326 0.226 0.435

(9.01$1076) (4.29$1008) (7.41$1019) (1.25$1041) (3.80$1006) (1.39$1009) (4.55$1026) (0.016) (1.10$1009)

LTA4-H 0.326 0.164 0.542 0.287 0.204 0.391

(2.96$1030) (0.056) (1.49$1012) (3.03$1020) (0.024) (4.95$1007)

negligible. However, the LTA4-H expression provides a good demonstration of how low-phenotypic correlations may misrepresent the true underlying relationships, since it provides strong evidence for correlations between genetic effects and environmental effects with arterial thrombosis, but exhibit different directions. In addition, due to the important relationship between inflammation and other prothrombotic mechanisms, such as coagulation and platelet aggregation, we have analyzed the correlation between LT traits and coagulation factors (i.e. Factors IX, VII, VIII, XI, XII and fibrinogen) and platelet function (i.e. platelet functional analyzer (PFA)-collagen/ADP and PFA-collagen/epinephrine) that were measured in the GAIT sample. No correlation was found between these parameters (data not shown). 4. Discussion The relatively high heritabilities that we found strongly indicate that genetic factors have a strong influence in determining LTrelated phenotypes, whereas household factors play a minor role. The genetic contribution that we found is limited to additive gene effects and excludes dominance and epistasis. Thus, our heritabilities are conservative estimates. In addition, it is important to note, that our results applied to the general Spanish population since we incorporate an ascertainment correction for the selection of a patient with thrombosis. Once we find the genetic determinant that influence thrombotic risk, they will explain thrombosis partly. These data suggest that it could be possible to use whole genome approaches to localize and characterize Quantitative Trait Loci (QTLs) that underlie the variability of LT-related phenotypes. In fact, recently, several polymorphisms have been identified in the LT-related genes that determine (in part) the LT levels in the general population [15]. Most of our knowledge of the genetic factors involved in the LT-biosynthetic pathway has been limited to association studies that employ caseecontrol designs that look at polymorphic variations in candidate genes. Although such studies provide important indirect evidence of genetic effects, they suffer from the inability to estimate reliably the relative importance of genetic factors within-population variation. Family-based studies do not suffer these problems. Moreover, the influence of these polymorphisms, if any, on plasma LT-related phenotypes is relatively small [15]. Hence, it is needed to perform a genomic search to localize genes that influence quantitative variation of LT-related phenotypes. Current results provide some new insights on the regulation of 5-LO pathway, since 5-LO, FLAP and LTC4-S exhibit significant environmental and genetic correlations. In contrast, LTA4-H, exhibit only environmental correlation with the other parameters. Evidence for strong genetic correlations among 5-LO, FLAP and LTC4-S indicate that there are sets of genes that jointly influence quantitative physiological variation of these traits.

FLAP

Arterial thrombosis

Venous thrombosis

0.594 (5.71$1086) 0.592 (6.54$1013) 0.598 (2.47$1014)

0.163 0.426 0.106 0.306 0.662 0.236 0.008 0.89e1 0.269

0.072 0.155 0.003 0.073 0.189 0.024 0.002 0.018 0.012

(0.071) (0.149) (0.398) (3.99$1005) (0.020) (0.045) (0.011) (4.79$1003) (0.021)

(0.440) (0.322) (0.980) (0.375) (0.242) (0.845) (0.992) (0.904) (0.916)

The 5-LO and FLAP are expressed exclusively in leukocytes and the LTB4 production by whole blood is therefore dependent on these cells. Production of LTB4 in response to A23187 exhibited a significant heritability although smaller than that of plasma concentration of LTB4. This is consistent with our previous report regarding prostaglandin (PG) E2 production by whole blood in response to A23187, which is also dependent on leukocytes. We found a heritability and the household component of A23187stimulated production of PGE2, either absolute or normalized for total leukocyte or monocyte densities, quantitatively quite similar to those reported for LTB4 [26]. The contribution of events in tissues to the circulating levels of LTB4, such as local inflammation, could account for the lack of correlation between LTB4-plasma levels and those of mRNA of its biosynthetic pathway enzymes in blood cells. Such tissue events are not discernible using the methods of our study. However, A23187-induced production of LTB4 did not correlated also with the expression of any of the mRNA. A23187stimulated levels of LTB4 were 1000-fold higher than those of plasma. Therefore, the potential contribution of tissues to the A23187-stimulated LTB4 levels is negligible. It suggests that mobilization of AAc by the upstream enzymes phospholipases could be the limiting step in the biosynthesis of LTs under basal and particularly under A23187-stimulated conditions. However, basal plasma levels of LTB4 exhibited a high heritability. This suggests that phospholipase expression and/or basal activity might be highly heritable also. It is important to note that the only common step in COX- and 5-LO pathways is the release of free AAc by phospholipases. In our previous report [26], we also found no correlation between prostanoid levels in response to A23187 and COX-isoenzyme, thromboxane A (TxA)-synthase and PGEsynthase-1. Moreover, since all whole blood samples from the previous study have been included in the present study, we have evaluated the correlation between TxA2 or PGE2 levels in calcium ionophore-stimulated whole blood samples and LTB4 levels. A significant correlation between LTB4 and either TxA2 or PGE2 was observed (not shown). Altogether, this suggest that calciumsensitive phospholipases are the limiting step in LT and prostanoid biosynthesis by activated leukocytes and that they are possible targets for pharmacological intervention in inflammation-related cardiovascular diseases. Of course, we have to recognize that these arguments are based on quite indirect data, and factors other than phospholipase activity could account for part in the absence of correlation between mRNA and LTB4 levels. LOs and COX activities can be regulated by redox state of the cells (e.g., HpETE levels) thus coordinate regulation could be a factor as well as phospholipase activity. It is important to note that we found a significant genetic correlation between FLAP and LTA4-H expression and arterial thrombosis. This provides strong evidence for significant pleiotropy underlying the covariation between these phenotypes (FLAP and LTA4-H expression) and arterial thrombosis risk.

134

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135

Our results of the LTA4-H expression demonstrate how lowphenotypic correlations may misrepresent the true underlying relationships, since it provides strong evidence for correlations between genetic effects and environmental effects with arterial thrombosis, but exhibit different directions. When such differences in sign occur, the phenotypic correlation is attenuated, although the underlying components suggest much stronger correlations. Our results are consistent with previous epidemiological results, where a four-SNP haplotypes in the FLAP gene has been associated with a greater risk of myocardial infarction and stroke [15] and with animal models, where the administration of FLAP antagonist reduces atherosclerosis [27]. However, FLAP expression did not correlate with venous thrombosis risk in this population. This difference between venous and arterial thrombosis regarding FLAP expression might be clinically relevant since a number of biological mechanisms are common to both, venous and arterial thrombosis [28]. During the past decade, the role of inflammation in the pathophysiology of arterial thrombosis has been elucidated. The data indicate that inflammation of the vessel wall initiates thrombus formation in an intact vein and that inflammation and coagulation systems have a common activation pathway [29]. Both 5-LO and FLAP are required for LTA4 synthesis [1,2] and regulation of FLAP expression may represent an important controller of LT biosynthesis. However, the role of LTs, and therefore FLAP, could be considered as pro-atherogenic and also linked with thrombosis associated with plaque rupture. The latter is likely more relevant in arterial than in the less stressed venous territory, where thrombosis could by mainly associated with coagulation and fibrinolysis alterations. Interestingly, oxidized LDL strongly upregulate FLAP expression in the macrophages cell line U937 [30], suggesting a mechanism for FLAP induction in macrophages during the atherosclerosis development. In addition, the lack of correlation between coagulation factors and platelet function parameters suggests that the association of 5-LO pathway parameters and arterial thrombosis could have underlying causes outside of coagulation or platelet function abnormalities (at least, regarding parameters measured in our sample). Further research is needed to determine whether these or other markers of inflammation could be an adjunct to the available diagnostic tools in the detection of thromboembolic diseases. Therefore, more extensive knowledge of these processes, and their underlying associated factors, would greatly improve our understanding of the common pathophysiological features of both venous and arterial thromboembolism. The fact that LTA4-H also correlated with arterial thrombosis specifically highlights the role of LTB4 and PMN in cardiovascular events. In conclusion, our results provide direct evidence of the major role of genetic factors in the determination of the phenotypic variability of leukotriene-related traits. Our study is a reasonable foundation for future studies that focus on understanding the mechanisms of genetic variation within the 5-LO pathway and their role in diseases where inflammation has been implicated. Regarding the role of 5-LO pathway in cardiovascular diseases, genetically-induced variations of these phenotypes might be important under pathological conditions. It is clear that the next step in the understanding of the genetic regulation of these traits is to localize the QTLs affecting their expression. Therefore, a genomic search to identify and assess the pathological implications of these QTLs is warranted. Funding This study was supported partially by grants No. PI-08/0420, PI-08/0756, SAF2008/01859, SAF2010/21392 and RECAVA-RD06/ 0014. J.M. Soria was supported by “Programa d’Estabilització

d’Investigadors de la Direcció d’Estrategia i Coordinació del Departament de Salut” (Generalitat de Catalunya). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflicts of interest There are not conflicts of interest. Acknowledgments We would like to acknowledge the advice and helpful discussion of Professor W.H. Stone. Also, we want to acknowledge the technical support of Antonio Cardenas, Luis Alegria, Joan Nicolau, Montserrat Font, Najia Khatab, Raquel Pérez and Biel Cuevas. Finally, we are indebted to all of the families who participated in the GAIT Project. References [1] Vila L. Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in atherosclerosis. Med Res Rev 2004;24:399e424. [2] Riccioni G, Zanasi A, Vitulano N, Mancini B, D’Orazio N. Leukotrienes in atherosclerosis: new target insights and future therapy perspectives. Mediators Inflamm 2009;2009. 737282. [3] Ford-Hutchinson AW, Bray MA, Doig MV, Shipley ME, Smith MJ, Leukotriene B. A potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 1980;286:264e5. [4] Lee E, Lindo T, Jackson N, et al. Reversal of human neutrophil survival by leukotriene B(4) receptor blockade and 5-lipoxygenase and 5-lipoxygenase activating protein inhibitors. Am J Respir Crit Care Med 1999;160:2079e85. [5] Qiu H, Gabrielsen A, Agardh HE, et al. Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability. Proc Natl Acad Sci U S A 2006;103:8161e6. [6] Aiello RJ, Bourassa P-A, Lindsey S, Weng W, Freeman A, Showell HJ. Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice. Arterioscler Thromb Vasc Biol 2002;22:443e9. [7] Zhao L, Moos MPW, Gräbner R, et al. The 5-lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med 2004; 10:966e73. [8] Bäck M, Bu D, Bränström R, Sheikine Y, Yan Z-Q, Hansson GK. Leukotriene B4 signaling through NF-kB-dependent BLT1 receptors on vascular smooth muscle cells in atherosclerosis and intimal hyperplasia. Proc Natl Acad Sci U S A 2005;102:17501e6. [9] Heller EA, Liu E, Tager AM, et al. Inhibition of atherogenesis in BLT1-deficient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruitment. Circulation 2005;112:578e86. [10] Houard X, Ollivier V, Louedec L, Michel JB, Bäck M. Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus. FASEB J 2009;23:1376e83. [11] In KH, Asano K, Beier D, et al. Naturally occurring mutations in the human 5lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription. J Clin Invest 1997;99:1130e7. [12] Dwyer JH, Allayee H, Dwyer KM, et al. Arachidonate 5-lipoxygenase promoter genotype, dietary arachidonic acid, and atherosclerosis. N Engl J Med 2004; 350:29e37. [13] Assimes TL, Knowles JW, Priest JR, et al. Common polymorphisms of ALOX5 and ALOX5AP and risk of coronary artery disease. Hum Genet 2008;123: 399e408. [14] González P, Reguero JR, Lozano I, Morís C, Coto E. A functional Sp1/Egr1tandem repeats polymorphism in the 5-lipoxygenase gene is not associated with myocardial infarction. Int J Immunogenet 2007;34:127e30. [15] Helgadottir A, Manolescu A, Thorleifsson G, et al. Stefansson K the gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat Genet 2004;36:233e9. [16] Bäck M. Inhibitors of the 5-lipoxygenase pathway in atherosclerosis. Curr Pharm Des 2009;15:3116e32. [17] Helgadottir A, Manolescu A, Helgason A, et al. A variant of the gene encoding leukotriene A4 hydrolase confers ethnicity-specific risk of myocardial infarction. Nat Genet 2006;38:68e74. [18] Yamada Y, Kato K, Oguri M, et al. Association of genetic variants with atherothrombotic cerebral infarction in Japanese individuals with metabolic syndrome. Int J Mol Med 2008;21:801e8. [19] Lovannisci DM, Lammer EJ, Steiner L, et al. Association between a leukotriene C4 synthase gene promoter polymorphism and coronary artery calcium in young women: the Muscatine study. Arterioscler Thromb Vasc Biol 2007;27: 394e9.

M. Camacho et al. / Atherosclerosis 224 (2012) 129e135 [20] Bevan S, Dichgans M, Wiechmann HE, Gschwendtner A, Meitinger T, Markus HS. Genetic variation in members of the leukotriene biosynthesis pathway confers an increased risk of ischemic stroke: a replication study in two independent populations. Stroke 2008;39:1109e14. [21] Freiberg JJ, Tybjaerg-Hansen A, Sillesen H, Jensen GB, Nordestgaard BG. Promotor polymorphisms in leukotriene C4 synthase and risk of ischemic cerebrovascular disease. Arterioscler Thromb Vasc Biol 2008;28:990e6. [22] Soria JM, Almasy L, Souto JC, et al. A genome search for genetic determinants that influence plasma fibrinogen levels. Arterioscler Thromb Vasc Biol 2005; 25:1287e92. [23] Buil A, Soria JM, Souto JC, et al. Protein C levels are regulated by a quantitative trait locus on chromosome 16: results from the genetic analysis of idiopathic thrombophilia (GAIT) project. Artherioscler Thromb Vasc Biol 2004;24: 1321e5. [24] Blangero J, Almasy L. Multipoint oligogenic linkage analysis of quantitative traits. Genet Epidemiol 1997;14:959e64.

135

[25] Almasy L, Blangero J. Multipoint quantitative trait linkage analysis in general pedigrees. Am J Hum Genet 1998;62:1198e211. [26] Vila L, Martínez-Pérez A, Camacho M, et al. Heritability of thromboxane A2 and prostaglandin E2 biosynthetic machinery in a spanish population. Arterioscler Thromb Vasc Biol 2010;30:128e34. [27] Jawien J, Gajda M, Olszanecki R, Korbut R. BAY X 1005 attenuates atherosclerosis in apoE/LDLRedouble knockout mice. J Physiol Pharmacol 2007;58:583e8. [28] Agnelli G, Becattini C. Venous thromboembolism and atherosclerosis: common denominators or different diseases? J Thromb Haemostasis 2006;4: 1886e90. [29] Poredos P, Jezovnik MK. The role of inflammation in venous thromboembolism and the link between arterial and venous thrombosis. Int Angiol 2007;26: 306e11. [30] Fair A, Pritchard Jr KA. Oxidized low density lipoprotein increases U937 cell 5lipoxygenase activity: induction of 5-lipoxygenase activating protein. Biochem Biophys Res Commun 1994;201:1014e20.