Smoking induces lipoprotein-associated phospholipase A2 in cardiovascular disease free adults: The ATTICA Study

Atherosclerosis 206 (2009) 303–308

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Smoking induces lipoprotein-associated phospholipase A2 in cardiovascular disease free adults: The ATTICA Study Alexandros D. Tselepis a,∗ , Demosthenes B. Panagiotakos b , Christos Pitsavos c , Constantinos C. Tellis a , Christina Chrysohoou c , Christodoulos Stefanadis c a b c

Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece Department of Nutrition Science - Dietetics, Harokopio University, Athens, Greece First Cardiology Clinic, School of Medicine, University of Athens, Athens, Greece

a r t i c l e

i n f o

Article history: Received 3 November 2008 Received in revised form 19 January 2009 Accepted 1 February 2009 Available online 21 February 2009 Keywords: Atherosclerosis Cardiovascular disease Inflammation Lp-PLA2 Smoking

a b s t r a c t Objectives: We studied the association of lipoprotein-associated phospholipase A2 (Lp-PLA2 ) mass and activity with various lifestyle, clinical and biochemical characteristics in cardiovascular disease (CVD) free adults. Background: Lipoprotein-associated phospholipase A2 is a novel biomarker of inflammation and risk for CVD. The Lp-PLA2 mass and activity are primarily influenced by the plasma levels of low-density lipoprotein (LDL), however, the influence of various lifestyle characteristics on Lp-PLA2 have not been adequately studied. Methods and results: In a random sub-sample of 186 subjects, 64 men (52 ± 13 years) and 122 women (48 ± 13 years) from the ATTICA Study (Greece), Lp-PLA2 activity and mass in total plasma as well as the enzyme activity and mass associated with high-density lipoprotein (HDL-Lp-PLA2 ) were determined using established methods. Several socio-demographic, lifestyle, clinical and biochemical parameters were assessed in all participants. Multiple linear regression analysis revealed that among the lifestyle characteristics, total plasma Lp-PLA2 activity and mass were positively and independently associated with current smoking (p = 0.02 and p = 0.05, respectively), as well as with exposure to second-hand smoke (p = 0.02 and p = 0.01, respectively). Furthermore, HDL-Lp-PLA2 activity and mass were inversely and independently associated with current smoking (p = 0.04 and p = 0.09, respectively). Conclusions: Smoking is associated with and might even induce an increase in proatherogenic total plasma Lp-PLA2 , but attenuates antiatherogenic HDL-Lp-PLA2 . These results further support the role of smoking as an important avoidable cause of CVD. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lipoprotein-associated phospholipase A2 (Lp-PLA2 ), also known as platelet-activating factor (PAF) acetylhydrolase, is a Ca2+ independent phospholipase A2 that degrades PAF and oxidized phospholipids [1]. Lp-PLA2 is associated mainly with apolipoprotein B (apoB)-containing lipoproteins and primarily with lowdensity lipoprotein (LDL), whereas a small proportion of circulating enzyme is also associated with high-density lipoprotein (HDL) [1,2]. In normolipidemic and dyslipidemic subjects, the majority of the LDL-associated Lp-PLA2 is bound to atherogenic small-dense LDL particles [2–5]. Furthermore, we have previously shown that enzyme activity is a marker of these particles in human plasma [5]. Lp-PLA2 is located with and highly expressed by macrophages

∗ Corresponding author. Tel.: +30 26510 98365; fax: +30 26510 98785. E-mail address: [email protected] (A.D. Tselepis). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.02.016

within the necrotic core and the fibrotic cap of advanced ruptureprone plaques [6,7]. Several epidemiologic studies support the proatherogenic role of total plasma Lp-PLA2 and show an independent association between Lp-PLA2 and cardiovascular disease (CVD) risk. In this regard, a meta-analysis showed that Lp-PLA2 is significantly associated with CVD, and the risk estimate appears to be relatively unaffected by adjustment for conventional CVD risk factors [8]. In contrast to the total plasma enzyme, which mainly represents the LDL-associated Lp-PLA2 , several lines of evidence suggest that HDLassociated Lp-PLA2 (HDL-Lp-PLA2 ) although present at low levels, may contribute to the antiatherogenic effects of this lipoprotein [1]. However, the clinical value of HDL-Lp-PLA2 as a potent inhibitor of the atherosclerotic process remains to be established. The “ATTICA” epidemiological study is a health and nutrition survey that has been carried out in the region of Attica, Greece [9,10]. Apparently healthy men and women, residents in the above area, were randomly selected to enrol into the study. In the present

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work we evaluated the associations of various lifestyle, clinical and biochemical characteristics with total plasma Lp-PLA2 activity or mass and HDL-associated enzyme activity or mass, in a random sub-sample of men and women from the ATTICA Study. 2. Methods 2.1. Study’s sample From May 2001 to December 2002, 4056 inhabitants from the region of Attica, Greece, were randomly selected to enrol into the “ATTICA” Study. Of them, 3042 agreed to participate (75% participation rate). For the purposes of the present work a random sub-sample of the ATTICA Study’s participants that were not on lipid lowering agents was selected. The sub-sample consisted of 186 subjects, 64 males (52 ± 13 years) and 122 females (48 ± 13 years). All participants were interviewed by trained personnel (cardiologists, general practitioners, dieticians and nurses) who used a standard questionnaire. From the design of the Study all participants had no history of CVD or any other atherosclerotic disease, as well as chronic viral infections. Moreover, participants did not have cold or flu, acute respiratory infection, dental problems or any type of surgery in the past week. The study was approved by the Medical Research Ethics Committee of First Cardiology Clinic, School of Medicine, University of Athens and was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association. 2.2. Lifestyle measurements According to the methodology applied in the original Study, smoking habits were assessed using a special questionnaire that included detailed information about cigarettes smoked per day, duration of smoking, types of cigarettes and exposure to secondhand smoke (i.e., passive smoking). The participants were classified as current smokers (i.e., those who were smoking on daily basis at the time of the interview, or those who stopped smoking within the last year), non-current smokers (i.e., those who had stopped smoking for at least 1 year) and never smokers. For a more detailed evaluation, pack-years of smoking were calculated as the product of the daily number of cigarettes smoked by the years of smoking. Exposure to second-hand smoke was evaluated through a special questionnaire. We inquired all participants: “Are you currently exposed to tobacco smoke from other people for more than 30 min per day?” We asked separately about two locations: home and workplace. Responses were categorized into three levels: no exposure to second-hand smoke, occasional exposure (i.e., up to 3 days per week, for at least 30 min per day), and regular exposure (i.e., more than 3 days per week, for at least 30 min per day). We also asked: “As an adult, how many years have you lived (i.e., home or workplace) with someone who has smoked regularly?” There was a high concordance between the answers of the participants and their relatives or accompanying persons about smoking habits (Kendall  in men = 0.82, p < 0.01; Kendall  in women = 0.93, p < 0.01). For the evaluation of physical activity status we used a translated version of the validated “International Physical Activity Questionnaire” (IPAQ), suitable for assessing population levels of self-reported physical activity [11]. The short version of IPAQ (9 items) that we used provided information on weekly time spent walking, in vigorous- and moderate-intensity and in sedentary activity. Participants were instructed to refer to all domains of physical activity. Intensity of physical activity was expressed as MET-min per week. Therefore, participants were classified as inactive, minimally active and HEPA active (health enhancing physical activity; a high active category), based on the following criteria: an individ-

ual, was classified as inactive, which is the lowest physical activity level, when no criteria were met to classify him or her in any of the other two categories; minimally active, which is the classification for sufficiently active, when any of the following three criteria were met: (a) 3 or more days of vigorous activity of at least 20 min per day, (b) 5 or more days of moderate-intensity activity or walking of at least 30 min per day, or (c) 5 or more days of any combination of walking, moderate-intensity or vigorous-intensity activities achieving of at least 600 MET-min/week; and HEPA active when any of the following criteria were met: (a) vigorous-intensity activity on at least 3 days achieving a minimum of at least 1500 METmin/week, or (b) 7 or more days of any combination of walking, moderate-intensity or vigorous intensity activity achieving a minimum of at least 3000 MET-min/week. Participants were instructed to report only episodes of activities of at least 10 min, since this is the minimum required to achieve health benefit. The evaluation of the nutritional habits was based on a validated food-frequency questionnaire (i.e., the EPIC-Greek questionnaire) that was kindly provided from the Nutrition Unit of Athens Medical School [12]. All participants were asked to report the daily or weekly average intake of several food items that they have consumed (during the last year). Alcohol consumption was measured in wineglasses (100 ml) and quantified by ethanol intake (grams per drink). One wineglass was equal to 12 g ethanol concentration. Based on the Mediterranean diet pyramid a diet score (range 0–55) was developed to describe a traditional dietary pattern [13,14] in order to evaluate overall dietary habits of the participants. In particular, for the consumption of items presumed to be close to this pattern (i.e., those suggested on daily basis or more than 4 servings per week) score 0 was assigned when a participant reported no consumption, 1 when reported consumption of 1–4 times/month, 2 for 5–8 times, 3 for 9–12 times/month, 4 for 13–18 times/month and 5 for more than 18 times/month. On the other hand, for the consumption of foods presumed to be away from this diet (like meat and meat products) the opposite scores were assigned (i.e., 0 when a participant reported almost daily consumption to 5 for rare or no consumption). Especially for alcohol score 5 was assigned for consumption of less than 3 wineglasses per day, score 0 for consumption of more than 7 wineglasses per day and scores 1–4 for consumption of 3, 4–5, 6 and 7 wineglasses per day. Higher values of this diet score indicate greater adherence to the Mediterranean diet. 2.3. Clinical and biochemical characteristics Resting arterial blood pressure was measured 3 times in the right arm, at the end of the physical examination with the subject in sitting position. Patients whose average blood pressure levels were greater or equal to 140/90 mmHg or were under anti-hypertensive medication were classified as having hypertension. Height and weight were recorded and body mass index (in kg/m2 ) was calculated. Fasting blood samples were collected from 08.00 to 10:00 h. The biochemical evaluation was carried out in the same laboratory that followed the criteria of the World Health Organization Reference Laboratories. Serum levels of total cholesterol, HDL-cholesterol, triglycerides and glucose were determined enzymatically on an automatic analyzer RA-1000 (Dade Behring, Marburg, Germany). LDL-cholesterol was calculated using the Friedewald formula (provided that triglyceride levels were less than 350 mg/dl). Serum levels of apolipoprotein A-I (apoA-I) and apoB were measured by immunonephelometry using a Beckman array analyzer (Beckman Instruments, Fullerton, CA, USA). The intra- and inter-assay coefficients of variation for all measured biochemical parameters did not exceed 4%. Diabetes mellitus was defined as a fasting serum glucose >125 mg/dl or the use of anti-diabetic medication.

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2.4. Measurement of Lp-PLA2 activity and mass Lp-PLA2 activity in total plasma and in apoB-depleted plasma, after the sedimentation of all apoB-containing lipoproteins with dextran sulfate–magnesium chloride (HDL-Lp-PLA2 activity), was determined by the trichloroacetic acid precipitation procedure using [3 H]-PAF (100 ␮mol/l final concentration) as a substrate [2,4]. Lp-PLA2 activity was expressed as nmol PAF degraded per min per ml of plasma. The Lp-PLA2 mass in total plasma as well as the HDL-Lp-PLA2 mass, were determined by a dual monoclonal antibody immunoassay standardized to recombinant Lp-PLA2 (PLAC test, diaDexus, Inc.), following the manufacturer instructions, as we previously described [5]. The Lp-PLA2 specific activity was calculated as the ratio of the enzyme activity to the enzyme mass (nmol per ng per min). 2.5. Statistical analysis Continuous variables are presented as mean values ± standard deviation (S.D.). Categorical variables are presented as frequencies. Associations between categorical variables were tested by the calculation of chi-squared test. Correlations between foodgroups intake and Lp-PLA2 levels were tested using the Spearman rho correlation coefficient. Multiple linear regression models were applied to test the association between the lifestyle characteristics and Lp-PLA2 levels, after controlling several potential confounders. Standardized residuals were used to test model’s goodness-of-fit. Normality of Lp-PLA2 levels was tested using the Kolmogorov–Smirnov criterion. All reported p-values are based on two-sided tests. SPSS 14 (SPSS Inc., Chicago, IL, USA) software was used for all the statistical calculations. 3. Results The distribution of several characteristics of the participants is shown in Table 1. Unadjusted analysis of the association of Lp-PLA2 activity or mass with various parameters presented in Table 1, revealed that Lp-PLA2 activity was positively correlated Table 1 Descriptive characteristics of the participants. Parameter Number of participants Age (years) Males, n (%) Body mass index (kg/m2 ) Hypertension, n (%) Diabetes, n (%) Total serum cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) Apolipoprotein B (mg/dl) Apolipoprotein A-I (mg/dl) Glucose (mg/dl) Total plasma Lp-PLA2 activity (nmol/(ml min)) Total plasma Lp-PLA2 mass (ng/ml) HDL-Lp-PLA2 activity (nmol/(ml min)) HDL-Lp-PLA2 mass (ng/ml)

186 49 ± 13 64 (34) 26.3 ± 4.5 58 (31) 19 (10) 199 ± 38 131 ± 35 53 ± 16 87 ± 18 107 ± 32 157 ± 20 100 ± 40 39 ± 10 279 ± 95 3.4 ± 1.3 79 ± 17

Physical activity status Sedentary, n (%) Minimally active, n (%) HEPA, n (%) Current smokers, n (%) Pack-years of smoking Non-current or never smokers, n (%) Second-hand smokers, n (%) Mediterranean diet score (0–55)

128 (69) 32 (17) 26 (14) 70 (38) 555 ± 541 79 (42) 37 (20) 25.4 ± 5.5

Data are presented as mean ± S.D. or frequencies.

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with age (r = 0.14, p = 0.05), pack-years of smoking (r = 0.25, p = 0.01), total serum cholesterol (r = 0.24, p = 0.001), LDL-cholesterol (r = 0.16, p = 0.05) and inversely correlated with HDL-cholesterol (r = −0.18, p = 0.01) and Mediterranean diet score (r = −0.13, p = 0.05). Similar findings were observed regarding Lp-PLA2 mass; particularly, Lp-PLA2 mass was positively correlated with age (r = 0.13, p = 0.05), pack-years of smoking (r = 0.27, p = 0.01), total serum cholesterol (r = 0.32, p < 0.001), LDL-cholesterol (r = 0.23, p = 0.01) and inversely correlated with HDL-cholesterol (r = −0.24, p = 0.001) and Mediterranean diet score (r = −0.16, p = 0.03). Moreover, LpPLA2 activity and mass were higher in males compared to females (43 ± 11 nmol/(ml min) vs. 36 ± 9 nmol/(ml min), p < 0.001, and 322 ± 150 ng/ml vs. 256 ± 128 ng/ml, p < 0.001, respectively). No correlation was found between hypertension and Lp-PLA2 activity or mass, a finding that accords to our previously published results showing that systolic blood pressure is a major determinant of plasma Lp-PLA2 activity only in patients with the metabolic syndrome suggesting that systolic blood pressure may have a cardinal role for plasma Lp-PLA2 levels only in coexistence with the rest of the metabolic syndrome features [15]. The HDL-Lp-PLA2 activity, was inversely correlated with body mass index (r = −0.19, p = 0.001) and pack-years of smoking (r = −0.24, p = 0.01), and positively correlated with Mediterranean diet score (r = 0.16, p = 0.03). Similarly with the aforementioned results we observed that HDL-Lp-PLA2 mass was inversely correlated with body mass index (r = −0.22, p = 0.002), pack-years of smoking (r = −0.20, p = 0.05) whereas it was positively correlated with Mediterranean diet score (r = 0.18, p = 0.04). Additionally, HDLLp-PLA2 activity and mass were lower in males compared to females (3.1 ± 0.8 nmol/(ml min) vs. 3.6 ± 0.9 nmol/(ml min), p = 0.002, and 74 ± 17 ng/ml vs. 82 ± 16 ng/ml, p = 0.004, respectively). Food group analysis, controlled for age, body mass index, smoking habits and physical activity status did not reveal any significant relationships between total plasma Lp-PLA2 activity or mass or HDL-Lp-PLA2 activity or mass and various foods or food groups consumed by the participants, as well as alcoholic beverages drinking. Despite the previous unadjusted associations, residual confounding may exist, thus we applied multi-adjusted regression analysis. This analysis revealed that lifestyle characteristics, LpPLA2 activity and mass were positively associated with current smoking and exposure to second-hand smoke in both current and non-current or never smokers. These associations were independent of various potential confounders presented in Table 2. By contrast, no associations were observed between Lp-PLA2 activity or mass and physical activity status or dietary habits (Table 2). Furthermore, a dose–response relationship was observed since pack-years of smoking were also independently, positively associated with Lp-PLA2 activity and mass (p < 0.01 for both correlations). As it is shown in Fig. 1A and B, the total plasma Lp-PLA2 activity and mass were significantly higher in current smokers as well as in second-hand smokers compared to non-current smokers. It should be noted that no association was found between pack-years of smoking and serum LDL-cholesterol or apoB levels (r = 0.13, p = 0.34 for both correlations). Importantly, the enzyme specific activity was significantly lower in current and second-hand smokers compared to non-current smokers (in nmol per ng per min, 0.11 ± 0.03 and 0.12 ± 0.04, respectively vs. 0.16 ± 0.02, p = 0.05 for both comparisons). Furthermore, the ratio of Lp-PLA2 mass to apoB levels was higher in active smokers and in second-hand smokers as compared to non-current smokers (2.8 ± 1.4 and 2.7 ± 1.1 vs. 2.0 ± 1.1, p = 0.03 and p = 0.05, respectively). As expected, other factors independently associated with Lp-PLA2 activity were male gender (p = 0.01), and LDL-cholesterol levels (p = 0.07) whereas Lp-PLA2 mass was positively associated only with LDL-cholesterol levels (p = 0.01). The interaction of effect of gender by smoking on Lp-PLA2 levels was also tested and found significant (p for interaction = 0.05 for mass

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Table 2 Association between plasma Lp-PLA2 activity and mass levels (dependent variables) and various characteristics of cardiovascular disease free adults. Model for Lp-PLA2 activity

B ± S.E.

Male vs. female gender Current smoking (yes/no) Second-hand smoking (yes/no) LDL-cholesterol (mg/dl) Hypertension (yes/no) Age (per 1 year) Body mass index (per 1 kg/m2 ) HEPA, minimally active vs. sedentary Mediterranean diet score (per 1 unit) Diabetes mellitus (yes/no)

5.70 5.14 4.72 0.05 2.43 0.06 −0.12 −1.02 0.048 −0.21

± ± ± ± ± ± ± ± ± ±

Model for Lp-PLA2 mass

B ± S.E.

Second-hand smoking (yes/no) LDL-cholesterol (mg/dl) Current smoking (yes/no) Male vs. female gender Hypertension (yes/no) Age (per 1 year) Diabetes mellitus (yes/no) Body mass index (per 1 kg/m2 ) HEPA, minimally active vs. sedentary Mediterranean diet score (per 1 unit)

90.62 1.10 61.80 47.57 44.7 −0.05 26.26 −1.01 4.09 −0.26

± ± ± ± ± ± ± ± ± ±

2.39 2.25 2.43 0.03 2.47 0.09 0.26 2.46 0.22 3.83

35.13 0.45 32.64 34.57 35.85 1.36 55.45 3.81 35.62 3.24

Table 3 Association between HDL-Lp-PLA2 activity and mass levels (dependent variables) and various characteristics of cardiovascular disease free adults.

p

Model for HDL-Lp-PLA2 activity

B ± S.E.

0.01 0.02 0.02 0.07 0.32 0.47 0.64 0.67 0.83 0.95

Body mass index (per 1 kg/m2 ) Current smoking (yes/no) Male vs. female gender LDL-cholesterol (mg/dl) Hypertension (yes/no) Mediterranean diet score (per 1 unit) Second-hand smoking (yes/no) Age (per 1 year) Diabetes mellitus (yes/no) HEPA, minimally active vs. sedentary

−0.03 −0.28 −0.26 −0.003 0.15 0.01 −0.11 0.004 −0.08 −0.03

p

Model for HDL-Lp-PLA2 mass

B ± S.E.

0.01 0.01 0.05 0.17 0.21 0.96 0.63 0.79 0.90 0.93

Body mass index (per 1 kg/m2 ) Male vs. female gender Current smoking (yes/no) LDL-cholesterol (mg/dl) Hypertension (yes/no) Mediterranean diet score (per 1 unit) Second-hand smoking (yes/no) Age (per 1 year) Diabetes mellitus (yes/no) HEPA, minimally active vs. sedentary

−0.78 −6.17 −5.50 −0.07 3.63 0.28 −2.86 0.07 −0.93 −0.43

and p = 0.01 for activity); suggesting that smoking (both active and passive) is associated with higher Lp-PLA2 levels in males compared to females (due to the small number of males and females these results should be generalized with conscious and therefore they are omitted). Regarding HDL-Lp-PLA2 multiple linear regression analysis revealed that among lifestyle characteristics, enzyme activity and mass were inversely and independently associated with current

Fig. 1. Total plasma Lp-PLA2 activity (A) and mass (B) according to the population smoking status. Enzymatic activity was determined by the trichloroacetic acid precipitation procedure and enzyme mass by use of a dual monoclonal antibody immunoassay. Values represent the mean ± S.D. *p < 0.05 compared with non-current smokers.

p ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

0.01 0.13 0.14 0.002 0.14 0.013 0.14 0.006 0.22 0.14

0.03 0.04 0.06 0.06 0.28 0.37 0.41 0.52 0.72 0.82 p

0.35 3.40 3.21 0.04 3.52 0.32 3.45 0.13 5.45 3.50

0.03 0.07 0.09 0.10 0.30 0.37 0.40 0.56 0.86 0.90

smoking (Table 3). Furthermore, a dose–response relationship was also observed between HDL-Lp-PLA2 activity and mass since they were inversely associated with pack-years of smoking (p = 0.03 for both correlations). By contrast, no significant association of HDLLp-PLA2 activity or mass was observed with second-hand smoking, in both smokers and non-current smokers. As it is shown in Fig. 2A and B the HDL-Lp-PLA2 activity and mass were lower in current smokers compared to non-current smokers, whereas no difference was observed in both parameters between non-current smokers

Fig. 2. HDL-Lp-PLA2 activity (A) and mass (B) according to the population smoking status. Enzymatic activity was determined by the trichloroacetic acid precipitation procedure and enzyme mass by use of a dual monoclonal antibody immunoassay. Values represent the mean ± S.D. *p < 0.05 compared with non-current smokers.

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and second-hand smokers. No associations were observed between HDL-Lp-PLA2 activity or mass and physical activity status or dietary habits (Table 3). It should be noted that pack-years of smoking were inversely correlated with serum HDL-cholesterol and apoA-I levels (r = −0.25, p = 0.01 for both comparisons). Importantly, the enzyme specific activity was significantly lower in current smokers compared to non-current smokers (in nmol per ng per min, 0.039 ± 0.01 vs. 0.045 ± 0.02, respectively, p = 0.05), whereas no difference was observed between second-hand and non-current smokers. Furthermore, the ratio of HDL-Lp-PLA2 mass to apoA-I levels was similar between current or second-hand smokers and non-current smokers (0.49 ± 0.6 or 0.49 ± 0.5 vs. 0.49 ± 0.7, respectively, p = 0.89 for both comparisons). Finally, as expected, HDL-Lp-PLA2 activity and mass were negatively associated with body mass index, male gender and LDL-cholesterol levels (Table 3). 4. Discussion The present work shows for the first time that the plasma Lp-PLA2 activity and mass are positively associated with active or second-hand smoking, among apparently healthy individuals without any clinical evidence of CVD. These associations were dose–response and independent from various lifestyle, biochemical and clinical characteristics of the participants. Epidemiological studies strongly support the assertion that active cigarette smoking is a major modifiable risk factor for CVD [16]. The mechanisms linking cigarette smoking to CVD include impairment of endothelial function elevation of various proinflammatory cytokines, increased expression of adhesion molecules on the surface of monocytes and endothelial cells, increase leukocyte–endothelial cell interaction, enhanced rate of transendothelial migration of monocytes, increased oxidative stress, modification of lipid profile, platelet dysfunction and alteration in prothrombotic and antithrombotic factors [17]. The relationship between cigarette smoking and various established risk factors for CVD has been investigated [18]. However, few data are available on the relationship between cigarette smoking and newly emerging risk factors for CVD. In this regard, it has been reported that cigarette smoking is strongly, positively and independently associated with C-reactive protein (CRP), fibrinogen and homocysteine [19]. A novel inflammatory marker or even an emerging risk factor for CVD is Lp-PLA2 . Indeed, a substantial body of peer-reviewed studies in Caucasian population has supported LpPLA2 as a cardiovascular risk marker independent of and additive to traditional risk factors. Increased Lp-PLA2 levels in plasma approximately double the risk for primary and secondary cardiovascular events [8]. Thus Lp-PLA2 in plasma should be viewed today as an important cardiovascular risk marker whose utility is as an adjunct to the major risk factors to adjust absolute risk status and thereby modify low-density lipoprotein cholesterol goals [20]. The present study shows that the Lp-PLA2 mass in plasma (which mainly reflects the LDL-associated enzyme) is positively associated with active smoking among healthy individuals, the current smokers exhibiting significantly higher enzyme mass compared to non-current smokers. One of the most important determinants of the plasma enzyme levels is the plasma LDL concentration [1]. According to our results, active smoking does not affect either LDL-cholesterol or apoB serum levels thus it is unlikely that the smoking-induced elevation in Lp-PLA2 mass is due to any increase in LDL plasma levels. Our data further demonstrated that the ratio of Lp-PLA2 mass to apoB levels is higher in current compared to non-current smokers thus suggesting that smoking may induce an increase of the Lp-PLA2 protein associated with LDL. It has been previously reported that cigarette smoking induces an elevation in the plasma levels of PAF or closely related lipids, in vivo [21]. Smoking one cigarette acutely increases the concentra-

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tion of oxidatively fragmented phospholipids in healthy adults, whereas 1 h after smoking, the concentration of these phospholipids returns to nearly the pre-smoking level [22]. Furthermore, cigarette smoke in hamsters induces the formation of neutrophiland platelet-stimulating lipids that most likely correspond to fragmented phosphatidylcholines [23]. PAF is the only proinflammatory mediator that stimulates the expression and secretion of Lp-PLA2 in differentiated monocyte-derived macrophages [24], the major source of enzyme in plasma [25]. Thus we may suggest that the increase in plasma Lp-PLA2 mass induced by cigarette smoking reported in the present study, could be at least partially attributed to the enhancement in enzyme production and secretion induced by PAF or related lipids formed in vivo in active smokers, a hypothesis that is currently under investigation in our laboratory. Previous studies have demonstrated that exposure of human plasma to a cigarette smoke extract inhibit Lp-PLA2 activity, in vitro [26]. In this regard it has been suggested that components of cigarette smoke may indirectly inhibit Lp-PLA2 by modifying the local environment on the surface of LDL on which the majority of plasma enzyme is accumulated [27]. Furthermore, Lp-PLA2 exhibits a modest sensitivity to gas-phase cigarette smoke, in vitro [27], which could be attributed to a direct effect of the increased oxidative stress induced by cigarette smoke on enzyme activity [28,29]. Indeed more recently it was shown that the enzyme inactivation induced in the presence of oxidative stress is irreversible and one of the primary targets of oxidation is the Met-117 residue of the enzyme protein. Furthermore, peroxynitrite is one of the species that can inactivate Lp-PLA2 inducing tyrosine nitration, possibly at residues Tyr-307 and Tyr-335 [30]. Consistent with the above data are the results of the present study showing that current smokers exhibit significantly lower Lp-PLA2 specific activity in plasma probably due to the smoking-induced partial inhibition of the enzyme activity. Consequently, the enhanced production and secretion of new active enzyme could counteract the smoking-induced partial enzyme inhibition, thus resulting in an overall increase in Lp-PLA2 activity in the plasma of current smokers. An important observation of the present study is that secondhand smoke also induces plasma Lp-PLA2 activity and mass to a similar extent as that observed in active smokers. Epidemiological studies have demonstrated that second-hand smoke significantly increases the risk of CVD [31]. Indeed, several studies have shown that endothelial and platelet function, inflammation, oxidative stress and atherosclerosis are exquisitely sensitive to the toxins of second-hand smoke [32]. Furthermore, passive smokers have increased levels of several biomarkers of CVD risk, including fibrinogen, homocysteine [33,34], white blood cell counts, oxidized LDL and CRP [34]. The present study is the first to show that, similarly to active smoking, passive smoking is also positively associated with plasma Lp-PLA2 activity and mass, thus providing an additional mechanism linking second-hand smoke with susceptibility to CVD. There is strong evidence that cigarette smoking induces a reduction in the plasma levels of the antiatherogenic HDL, and it may represent one of the important mechanisms by which cigarette smoking increases the CVD risk [18]. The inverse correlation between pack-years of smoking and serum HDL-cholesterol and apoA-I levels found in the present study, are in accordance to the above results. An important role in the antiatherogenic effects of HDL may be played by the HDL-Lp-PLA2 . The antiatherogenic role of HDL-Lp-PLA2 has been supported by studies in vitro, as well as by in vivo studies in animal models [35]. On a clinical basis of view, the decrease in the ratio of HDL-Lp-PLA2 to total plasma enzyme or to LDL-Lp-PLA2 observed in patients with dyslipidemias, may be useful as a potential marker of atherogenicity in these subjects [4,36,37]. Furthermore, HDL-Lp-PLA2 activity is inversely associated with the degree of insulin resistance, as it

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was assessed by the HOMA index (a marker of insulin resistance) in patients with the metabolic syndrome [15]. The present study shows for the first time that the HDL-Lp-PLA2 mass is inversely associated with current smoking. Since the ratio of HDL-Lp-PLA2 mass to apoA-I is not influenced by smoking it is suggested that the main mechanism for the reduction of HDL-Lp-PLA2 mass in our population is the smoking-induced reduction of plasma HDL levels. Furthermore, current smokers exhibit low HDL-LpPLA2 activity and low enzyme specific activity a result of the low HDL-Lp-PLA2 mass and smoking-induced enzyme partial inactivation. The present study has the following limitations: the number of enrolled participants may be considered small for epidemiologic investigations; however, the observed statistical power in the presented models varied from 65% to 88% (at 0.05 level of significance). Smoking habits were assessed through a questionnaire and not using an objective measurement (e.g., serum cotinine levels); however, this procedure is considered reliable in epidemiologic studies). Because of the cross-sectional design of the study it cannot establish causal relations, but only generate a hypothesis for the association of the investigated characteristics with Lp-PLA2 levels. Finally, although a gender difference was observed regarding Lp-PLA2 levels as well as smoking habits, it is hard to split the analysis by gender due to the small number of participants in each group. In summary, the present study shows for the first time that current smoking as well as exposure to second-hand smoke is associated with an increase of total plasma Lp-PLA2 , the novel inflammatory marker of CVD, and attenuates the anti-inflammatory HDL-Lp-PLA2 . These findings lend support to existing evidence that both active and passive smoking have an important effect on susceptibility to atherosclerotic disease and further support the role of smoking as an important avoidable cause of CVD. Conflict of interest No conflict of interest or any financial disclosure exists. Acknowledgements The ATTICA Study is supported by research grants from the Hellenic Society of Cardiology (HCS2002). References [1] Tselepis AD, Chapman MJ. Inflammation, bioactive lipids and atherosclerosis: potential roles of a lipoprotein-associated phospholipase A2 , platelet activating factor-acetylhydrolase. Atherosclerosis Suppl 2002;3:57–68. [2] Tselepis AD, Dentan C, Karabina S-A, et al. PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VLDL-1 in human plasma: catalytic characteristics and relation to the monocyte-derived enzyme. Arterioscler Thromb Vasc Biol 1995;15:1764–73. [3] Karabina S-A, Liapikos TA, Grekas G, et al. Distribution of PAF-acetylhydrolase activity in human plasma low density lipoprotein subfractions. Biochim Biophys Acta 1994;1213:34–8. [4] Tsimihodimos V, Karabina S-A, Tambaki AP, et al. Altered distribution of platelet activating factor-acetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia. J Lipid Res 2002;43:256–63. [5] Gazi I, Lourida ES, Filippatos T, et al. Lipoprotein-associated phospholipase A2 activity is a marker of small, dense LDL particles in human plasma. Clin Chem 2005;51:2264–73. [6] Hakkinen T, Luoma JS, Hiltunen MO, et al. Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1999;19:2909–17.

[7] Kolodgie FD, Burke AP, Skorija KS, et al. Lipoprotein-associated phospholipase A2 protein expression in the natural progression of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 2006;26:2523–9. [8] Garza CAMV, McConnell JP, Somers VK, et al. Association between lipoproteinassociated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc 2007;82:159–65. [9] Pitsavos C, Panagiotakos DB, Chrysohoou C, Stefanadis C. Epidemiology of cardiovascular risk factors in Greece: aims, design and baseline characteristics of the ATTICA study. BMC Public Health 2003;3:32. [10] Panagiotakos DB, Pitsavos C, Chrysohoou C, et al. Status and management of blood lipids in Greek adults and their relation to socio-demographic, lifestyle and dietary factors: the ATTICA Study. Blood lipids distribution in Greece. Atherosclerosis 2004;173:353–61. [11] Rizos E, Tambaki AP, Gazi I, Tselepis AD, Elisaf M. Lipoprotein-associated PAF-acetylhydrolase activity in subjects with the metabolic syndrome. Prostaglandins Leukot Essent Fatty Acids 2005;72:203–9. [12] Ockene IS, Miller NH. Cigarette smoking, cardiovascular disease, and stroke: a statement for healthcare professionals from the American Heart Association: American Heart Association Task Force on Risk Reduction. Circulation 1997;96:3243–7. [13] Ambrose JA, Barua RS. The pathophysiology of cigarette smoking and cardiovascular disease. An Update. J Am Coll Cardiol 2004;43:1731–7. [14] Muscat JE, Harris RE, Haley NJ, Wynder EL. Cigarette smoking and plasma cholesterol. Am Heart J 1991;121:141–7. [15] Bazzano LA, He J, Muntner P, et al. Relationship between cigarette smoking and novel risk factors for cardiovascular disease in the United States. Ann Intern Med 2003;138:891–7. [16] Corson MA, Jones PH, Davidson MH. Review of the evidence for the clinical utility of lipoprotein-associated phospholipase A2 as a cardiovascular risk marker. Am J Cardiol 2008;101:41F–50F. [17] Imaizumi T, Satoh K, Yoshida H, et al. Effect of cigarette smoking on the levels of platelet-activating factor-like lipid(s) in plasma lipoproteins. Atherosclerosis 1991;87:47–55. [18] Frey B, Haupt R, Alms S, et al. Increase in fragmented phosphatidylcholine in blood plasma by oxidative stress. J Lipid Res 2000;41:1145–53. [19] Lehr HA, Weyrich AS, Saetzler RK, et al. Vitamin C blocks inflammatory platelet-activating factor mimetics created by cigarette smoking. J Clin Invest 1997;99:2358–64. [20] Cao Y, Stafforini DM, Zimmerman GA, McIntyre TM, Prescott SM. Expression of plasma platelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of inflammation. J Biol Chem 1998;273:4012–20. [21] Stafforini DM, Elstad MR, McIntyre TM, Zimmerman GA, Prescott SM. Human macrophages secrete platelet-activating factor acetylhydrolase. J Biol Chem 1990;265:9682–7. [22] Miyaura S, Eguchi H, Johnston JM. Effect of cigarette smoke extract on the metabolism of the proinflammatory autacoid, platelet-activating factor. Circ Res 1992;70:341–7. [23] Bielicki JK, Knoff LJ, Tribble DL, Forte TM. Relative sensitivities of plasma lecithin: cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure. Atherosclerosis 2001;155:71–8. [24] Frei B, Forte TM, Ames BN, Cross CE. Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma. Biochem J 1991;277:133–8. [25] Ambrosio G, Oriente A, Napoli C, et al. Oxygen radicals inhibit human plasma acetylhydrolase, the enzyme that catabolizes platelet-activating factor. J Clin Invest 1994;93:2408–16. [26] MacRitchie AN, Gardner AA, Prescott SM, Stafforini DM. Molecular basis for susceptibility of plasma platelet-activating factor acetylhydrolase to oxidative inactivation. FASEB J 2007;21:1164–76. [27] He J, Vupputuri S, Allen K, et al. Passive smoking and the risk of coronary heart disease: a meta-analysis of epidemiologic studies. N Engl J Med 1999;340:920–6. [28] Barnoya J, Glantz SA. Cardiovascular effects of secondhand smoke. Nearly as large as smoking. Circulation 2005;111:2684–98. [29] Venn A, Britton J. Exposure to secondhand smoke and biomarkers of cardiovascular disease risk in never-smoking adults. Circulation 2007;115:990–5. [30] Panagiotakos DB, Pitsavos C, Chrysohoou C, et al. Effect of exposure to second hand smoke on inflammation: the ATTICA study. Am J Med 2004;116:145–50. [31] Stafforini D. Biology of platelet-activating factor acetylhydrolase (PAFAH lipoprotein associated phospholipase A(2)). Cardiovasc Drugs Ther 2009;23:73–83. [32] Tsimihodimos V, Kakafika A, Tambaki AP, et al. Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoBcontaining lipoproteins. J Lipid Res 2003;44:927–34. [33] Tsimihodimos V, Karabina SA, Tambaki AP, et al. Atorvastatin preferentially reduces LDL-associated platelet-activating factor acetylhydrolase activity in dyslipidemias of type IIA and type IIB. Arterioscler Thromb Vasc Biol 2002;22:306–11.