Atherosclerosis 234 (2014) 34e41

Contents lists available at ScienceDirect

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

Association of Lp-PLA2 with digital reactive hyperemia, coronary flow reserve, carotid atherosclerosis and arterial stiffness in coronary artery disease Ignatios Ikonomidis*, Nikolaos N.P. Kadoglou, Vlassis Tritakis, Ioannis Paraskevaidis, Kleanthi Dimas, Paraskevi Trivilou, Ioannis Papadakis, Stavros Tzortzis, Helen Triantafyllidi, John Parissis, Maria Anastasiou-Nana, John Lekakis Second Department of Cardiology, Athens University Medical School, Attikon Hospital, Athens, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 October 2013 Received in revised form 1 February 2014 Accepted 7 February 2014 Available online 18 February 2014

Background: Lipoprotein-associated Phospholipase A2 (Lp-PLA2), has a powerful inflammatory and atherogenic action in the vascular wall and is an independent marker of poor prognosis in coronary artery disease (CAD). We investigate the association of Lp-PLA2 with markers of vascular dysfunction and atherosclerosis with proven prognostic value in CAD. Methods: In 111 patients with angiographically documented chronic CAD, we measured 1) carotid intima-media thickness (CIMT), 2) reactive hyperemia using fingertip peripheral arterial tonometry (RHPAT), 3) coronary flow reserve (CFR), by Doppler echocardiography 4) pulse wave velocity (PWV) and 5) blood levels of Lp-PLA2. Results: Patients with Lp-PLA2 concentration >234.5 ng/ml (50th percentile) had higher CIMT (1.44
0.07 vs. 1.06
0.06 mm), PWV (11.0
2.36 vs. 9.7
2.38 m/s) and lower RH-PAT(1.24
0.25 vs. 1.51
0.53) and CFR (2.39
0.75 vs. 2.9
0.86) compared to those with lower Lp-PLA (p < 0.05 for all comparisons). Lp-PLA2 was positively associated with CIMT (regression coefficient b: 0.30 per unit of LpPLA2, p ¼ 0.02), PWV (b:0.201, p ¼ 0.04) and inversely with RHI-PAT (b: 0.371, p < 0.001) and CFR (b: 0.32, p ¼ 0.002). In multivariate analysis, Lp-PLA2 was an independent determinant of RHI-PAT, CFR, CIMT and PWV in a model including age, sex, smoking, diabetes, dyslipidemia and hypertension (p < 0.05 for all vascular markers). Lp-PLA2, RHI-PAT and CFR were independent predictors of cardiac events during a 3-year follow-up. Conclusions: Elevated Lp-PLA2 concentration is related with endothelial dysfunction, carotid atherosclerosis, impaired coronary flow reserve and increased arterial stiffness and adverse outcome in CAD patients. These findings suggest that the prognostic role of Lp-PLA2 in chronic CAD may be explained by a generalized detrimental effect of this lipase on endothelial function and arterial wall properties. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lp-PLA2 Coronary artery disease Reactive hyperemia index Coronary flow reserve Carotid intima-media thickness Pulse wave velocity Prognosis

1. Introduction The lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), consists a 50 kDa lipase with inflammatory and atherogenic action and an independent prognostic value in primary and secondary prevention [1,2]. It is produced primarily by * Corresponding author. Second University Cardiology Department, Attikon University Hospital, Rimini 1, Haidari, 12462 Athens, Greece. Tel.: þ30 210 5832187; fax: þ30 210 5832351. E-mail addresses: [email protected], [email protected] (I. Ikonomidis). http://dx.doi.org/10.1016/j.atherosclerosis.2014.02.004 0021-9150/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

macrophages, but also by monocytes, T-lymphocytes, and mast cells [1]. Within the vessel wall, the interaction between oxidatively-modified LDL and Lp-PLA2, generates oxidized fatty acids (OxFA) and lysophosphatidylcholine (Lyso-PC) which are powerful inflammatory and atherogenic factors [3]. Increased expression of Lp-PLA2 has been observed in atherosclerotic lesions in animal and human models [4,5]. Furthermore, the circulating level of Lp-PLA2 has been proven an independent predictor for the development of coronary artery disease (CAD) [6]. However the association of Lp-PLA2 with impaired endothelial function, coronary microcirculation and arterial elasticity in CAD has not been fully investigated.

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41

The measurement of peripheral vasodilator response using fingertip Peripheral Arterial Tonometry (PAT) provides a useful method for assessing arterial endothelial function [7,8]. Although PAT signal is modulated by various local, systemic, and environmental factors, this parameter is predominantly determined by the bioavailability of NO [9]. Previous studies have shown an independent association of reactive hyperemia (RH-PAT) index with coronary endothelial function [10] and cardiovascular risk in patients with CAD [11]. Coronary flow reserve assessed by Doppler echocardiography (CFR) is a reliable, non-invasive method to identify epicardial coronary patency as well as coronary microcirculatory integrity [12e 15] .The scaling values of decreasing CFR constitute a comprehensive indicator of cardiovascular risk even in the presence of critical epicardial coronary stenosis [13], This is because it reflects the extent of coronary microcirculatory dysfunction and is related with left ventricular diastolic dysfunction [14,15]. Other valid markers of atherosclerosis are carotid intima-media thickness (CIMT) [16] and arterial stiffness quantified by pulse wave velocity (PWV) [17]. These vascular markers are interrelated [18] and associated with the extent of CAD [19], left ventricular diastolic dysfunction [15] and the risk for acute coronary event [20e 27], in the secondary prevention settings. Thus Lp-PLA2 and vascular markers are may offer further risk stratification and reclassify patients with overt cardiovascular disease to a higher risk level suggestive of the need of an intensified treatment [1,2,21e27]. To this extent, an ongoing multicenter trial aims to prove an improved prognosis after inhibition of Lp-PLA2 by darapladib in patients with chronic CAD [22]. In the present study we hypothesized a generalized detrimental effect of Lp-PLA2 on endothelial function as well as on the arterial wall properties across various vascular beds which would explain the independent prognostic value of this lipase in CAD. Thus, we examined the association of circulating Lp-PLA2 with a cluster of established markers of vascular function with an independent prognostic value in CAD namely RH-RAT, CFR, CIMT and PWV. 2. Methods

35

Table 1 Clinical, biochemical and vascular markers of the study population. Variables Clinical Age (y) Gender (males), n (%) Hypertension, n (%) DM, n (%) Dyslipidemia, n (%) Smoking, n (%) LAD, n (%) Multi-vessel, n (%) FH of CAD, n (%) SBP DBP Biochemical Chol (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) FPG (mg/dl) CRP (mg/L) Lp-PLA2 (ng/ml) Medications ASA Nitrates ACEIs/ARBS CCBs Statins b-blockers Vascular markers CFR RHI-PAT CIMT (mm) PWV (m/s)

Values 60
9 95 (85.2) 64 (57.6) 41 (36.9) 105 (94.5) 46 (41.4) 74 (66.7) 62 (55.8) 39 (35.1) 125
18 76
10 193
46 148
65 40
11 132
37 108
36 3.1
1.4 235
93 111 (100) 61 (55) 94 (84.6) 5 (4.5) 105 (94.5) 95 (85.5) 2.74
0.96 1.37
0.43 1.25
0.6 10.31
2.31

FH, family history; CAD, coronary artery disease; DM, diabetes mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; ASA, acetyl salicylic acid; ACEIs, angiotensin converting enzyme inhibitors; ARBs, angiotensin receptors blockers; CCBs, calcium channel blockers; Chol, total cholesterol; TG, triglycerides; LDL, Low density; HDL high density lipoprotein; FPG, fasting plasma glucose; CRP, C-reactive protein; Lp-PLA2, lipoproteinphospholipase A2; Patients with multivessel coronary artery disease before revascularisation; CFR, coronary flow reserve; RHI-PAT, reactive hyperemia index; CIMT, carotid intimaemedia thickness; PWV, pulse wave velocity.

2.1. Study population We enrolled 111 patients (85.2% men, mean age 60
9 years) with a) exercise- and/or stress-related angina b) evidence of reversible ischemia during stress echocardiography or thallium scintigraphy c) stenosis of 50% in the left main coronary artery and or 70% in one or several of the major coronary arteries within a year before inclusion in the study as defined in the ESC guidelines [23] (Table 1). All participants attended our preventive medicine laboratory. Using valid questionnaire, we recorded pharmaceutical regimens and other cardiovascular risk factors (smoking, hypertension, diabetes mellitus, dyslipidemia, family history of CAD). Exclusion criteria were: the presence of acute infection, malignancy, chronic heart failure (class NYHA III and IV), chronic obstructive pulmonary disease, recent major surgery, and severe chronic auto-immune diseases, liver and renal impairment. We also excluded patients with recent (within 6 months) acute cardiovascular events. Blood sampling for measurement of Lp-PLA2 was performed on the morning before we performed echocardiography and vascular tests in all patients. Patients were followed-up for death, new myocardial infarction with or without ST segment elevation with elevated troponin as defined according to current guidelines [24] for a period of 3 years. The study protocol was approved by the Local Ethics Committee, conducted in compliance with the Declaration of Helsinki and written informed consent was obtained from all patients before study entrance.

2.2. Pulse amplitude tonometry Measurement of peripheral vasodilator response with fingertip Peripheral Arterial Tonometry (PAT) technology (EndoPAT, Itamar Medical, Caesarea, Israel) is emerging as a useful method for assessing vascular function. The PAT device consists of two fingermounted probes, which include a system of inflatable latex aircushions within a rigid external case. A blood pressure cuff is placed on one upper arm (study arm), while the contralateral arm serves as a control (control arm) [8]. The reactive hyperemia Peripheral Arterial Tonometry (RH-PAT) index is calculated as the ratio of the average amplitude of the PAT signal over a 1-min time interval starting 1 min after cuff deflation divided by the average amplitude of the PAT signal of a 3.5-min time period before cuff inflation (baseline) [10] An RHI < 1.35 has been related with impaired coronary endothelial function [10] All studies were stored digitally and were analyzed by personnel blinded to clinical and laboratory data, using a computerized station. 2.3. Echocardiography Studies were conducted using a Vivid 7 (GE Medical Systems, Horten Norway) phased array ultrasound system using second harmonic imaging. All studies were stored digitally and were

36

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41

analyzed by two observers blinded to clinical and laboratory data, using a computerized station (Echopac GE, Horten Norway). All patients had adequate quality of images for analysis.

Table 2 Clinical and biochemical parameters of the study population divided by the median values of Lp-PLA2.

2.4. Coronary flow reserve (CFR) We assessed transthoracic Doppler Echocardiographic-derived coronary flow reserve was by obtaining the color-guided pulsewave Doppler signals. In the long axis apical projections using a 7 MHz transducer, we recorded the maximal velocity in the distal LAD at baseline and during hyperemic conditions after the intravenous administration of adenosine (0.14 mg/kg/min) [12e15] for 3 min. Measurements of three cardiac cycles were averaged. CFR was calculated as the ratio of hyperemic to resting maximal diastolic velocity. 2.5. Carotid intima media thickness (CIMT) CIMT was measured in three paired segments of both carotid arteries from a fixed 60 transducer angle using B-mode ultrasound imaging (7.0e10 MHz, linear array transducer). Measurements were made at the level of common carotid artery (defined as the segment 1 cm proximal to carotid bulb), at the level of carotid bulb and at the level of the internal carotid artery (defined as 1 cm-long arterial segment distal to the carotid bifurcation) using an automated tracing system (Vivid 7 GE Medical Systems, Horten Norway) [15]. In each segment, three measurements of the maximal CIMT in the far wall were averaged. The average CIMT of all six segments was calculated. Focal thickening at least 50% greater than that of the surrounding vessel wall was defined as a discrete plaque. 2.6. PWV measurement The carotid-femoral PWV was assessed by measuring the pulse transit time and the distance traveled between the two recording sites (PWV [m/s] ¼ travel distance [m]/transit time [s]). For pulse wave recording we used a validated noninvasive device (Complior SPÒ, Alam Medical, France) with capability of online wave recording. A simultaneous recording was performed by two pressure-sensitive transducers of two different pulse waves based over the right common carotid artery and the right femoral artery, respectively. Measurement of the distance between the transducers over the body surface allowed obtaining PWV. Measurements were performed by a single observer, blinded to clinical and laboratory data, and the whole procedure has been internally validated in our laboratory [15,17]. 2.7. Lp-PLA2 levels Serum levels of Lp-PLA2 were measured in our biochemistry laboratory with a commercially available enzyme-linked immunoassay (ELISA) (PLAC test, diaDexus, Inc, San Francisco, CA) with minimum detection limit of 0.34 ng/ml. The inter- and intra assay variations were 234.5 ng/ml (N ¼ 55)

p

60
11 40 (71.42) 37 (66.07) 22 (39.29) 51 (91.07) 26 (46.43) 56 (100%) 30 (53.5) 47 (83.9) 3 (5.3) 53 (94.6) 47 (83.9) 42 (75) 22 (39) 28 (50%) 124
17 76
10 199
40 143
55 41
13 137
33 117
41 2.61
1.16 164.28
41.60 1.51
0.53 2.9
0.86 1.06
0.6 9.7
2.38

60
9 55 (94.55) 27 (49.09) 19 (34.55) 54 (98.18) 20 (36.36) 55 (100%) 31 (56.3) 47 (85.4) 2 (3.6) 52 (94.5) 48 (87.2) 32 (58.18) 40 (72) 42 (76) 127
19 77
10 197
44 144
72 40
8 131
39 106
15 3.43
1.52 298.04
76.89 1.24
0.25 2.39
0.75 1.44
0.7 11.0
2.36

0.903 0.012 0.359 0.764 0.802 0.660 1 0.891 0.901 0.901 0.982 0.898 0.279 0.030 0.010 0.306 0.697 0.743 0.930 0.629 0.440 0.079 0.399 0.003 0.012 0.009 0.03

Lp-PLA2, lipoprotein-phospholipase A2; SBP, systolic BP; DBP, diastolic BP; TChol, total cholesterol; TG, triglycerides; FPG, fasting plasma glucose; CRP, C-reactive protein; RHI-PAT, reactive hyperemia index by Peripheral Arterial Tonometry; CFR, coronary flow reserve; CIMT, carotid intima-media thickness; PWV, pulse wave velocity.

were tested by the KolmogoroveSmirnof test to assess the normality of distribution. As Lp-PLA2, RH-PAT, CFR, CIMT, PWV did not have a normal distribution they were transformed into ranks for further analysis [24]. After transformation into ranks, linear regression analysis was performed to calculate the unstandardized regression coefficient b and standard error (SE) per unit of Lp-PLA2 to assess correlations between variables of interest. Multiple linear regression analysis was performed to examine predictors of each out of four vascular parameters (RH-PAT, CFR, CIMT and PWV) in our cohort. Finally, patients were categorized into equal subgroups, according to the median values of Lp-PLA2 and the measured vascular markers in our study cohort and the corresponding KaplaneMeier survival curves were constructed and compared using the logerank test. Multivariate logistic regression analysis was performed to calculate the odds ratio (OR) and 95% confidence intervals (CI) of the median Lp-PLA2 value for the median value of vascular markers after adjustment for various confounders. Multivariate Cox regression analysis was performed to calculate the hazard ratio (HR) and 95% confidence intervals (CI) of the median Lp-PLA2 and median value of vascular markers for the prediction of cardiac events after adjustment for various confounders.

2.8. Statistical analysis

3. Results

All variables are expressed as mean
SD or medians. Statistical analysis was performed using SPSS 17.0 statistical software package (SPSS, Chicago, IL, USA). Mean values of continuous variables were compared between groups using unpaired Student’s t-test. Categorical data were analyzed using the standard c2 test. Variables

3.1. Study population characteristics Clinical and biochemical characteristics of our study population are presented in Table 1. The mean values of the vascular parameters and the pharmaceutical regimen of the study cohort are shown

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41

in Table 1. Patients were categorized according to the median value of Lp-PLA2 (234.5 ng/ml) of our cohort which is similar to the value proposed to use as a clinical decision threshold (235 ng/ml) based on the 50th percentile of larger cohorts [25]. The subgroup of patients with Lp-PLA2 concentrations >234.5 ng/ml presented reduced values of RH-PAT and CFR as well as increased CIMT and PWV (Table 2, p < 0.05). Additionally, those with Lp-PLA2 concentrations >234.5 ng/ml had higher prevalence of multivessel CAD and carotid plaques (Table 2, p < 0.05). The age, sex, blood pressure, lipids and medication were similar between those with low and high Lp-PLA2 concentrations (p > 0.05). The patients with carotid plaques (n ¼ 70 (63%)) had higher levels of Lp-PLA2 than those without (271.21
79.12 ng/ml vs. 164.21
62.29 ng/ml p < 0.001). Additionally, patients with multivessel CAD (n ¼ 62) had higher Lp-PLA2 levels than patients with single vessel disease (259
100 ng/ml vs. 218
72 ng/ml p ¼ 0.03). 3.2. Interrelation between vascular markers Reduced CFR was associated with elevated systolic BP (regression coefficient b: 0.45, SE:0.15, per unit p ¼ 0.003) and reduced RHI-PAT (b: 0.201, SE:0.12, per unit, p ¼ 0.04). Reduced RHI-PAT was also related with increased CIMT (b: 0.20, SE:0.086, per unit p ¼ 0.025). PWV was related with hypertension (b: 23.6, SE:6.6 p ¼ 0.001), dyslipidemia (b: 18, SE:8.6, p ¼ 0.04), age (b: 1.5, SE:0.3, p < 0.001), and systolic BP (b: 0.54, SE:0.15, p ¼ 0.001) and had a borderline association with CFR (b: 0.19, SE:0.12, per unit, p ¼ 0.05).

37

not shown). In multiple regression analysis we examined the above correlations after adjustment for age, male sex, traditional cardiovascular risk factors, namely diabetes, hypertension, smoking and dyslipidemia as well as multivessel CAD to adjust for the extent of disease (model 1). As shown in Table 3, Lp-PLA2 remained an independent predictor of RHI-PAT, CFR, CIMT and PWV (p < 0.05 for all associations). We also performed additional adjustments for other potential confounders such as, LDL, systolic blood pressure, glucose levels in addition to age, sex and smoking in a different model to avoid co-linearity. Again Lp-PLA2 remained an independent predictor of the examined vascular markers (Table 3, model 2). Inclusion of statins instead of dyslipidemia or LDL in both models did not change the regression coefficients and SE of Lp-PLA2 (data not shown). Similarly inclusion of b-blockers, nitrates ACE/ ARBs in the model did not change the regression coefficients and SE of Lp-PLA2 (data not shown). Patients were also categorized into equal subgroups, according to the median values of measured vascular markers in our study cohort (RH-PAT  1.26, CFR  2.54, CIMT  2 mm, PWV  10 m/s). By logistic regression analysis an LpPLA2 > 234.5 ng/dl was good predictor of RH-PAT < 1.26 (OR:3.5, 95%CI: 1.5e8.3, p ¼ 0.02), CFR < 2.54, (OR:2.57 95%CI:1.01e5.5 p ¼ 0.03), CIMT > 1.2 mm (OR:4.1 95%CI:1.1e10.3, p ¼ 0.003), PWV > 10 m/s (OR:2.5, 95% CI:1.01e5.5, p ¼ 0.03), presence of carotid plaques (OR:2.6, 95%CI:1.4e6.5 p ¼ 0.035) and multivessel CAD (OR:2.1 95%CI:1.01e6.5 p ¼ 0.045) after adjustment for age, sex, and traditional cardiovascular risk factors, namely diabetes, hypertension, smoking and dyslipidemia and medication. 3.4. Associations between Lp-PLA2, vascular markers and cardiac events

3.3. Associations between Lp-PLA2 and vascular markers By regression analysis, elevated Lp-PLA2 were related with reduced RH-PAT (regression coefficient b per unit of LpPLA2: 0.371 SE:0.098, p < 0.001) and CFR (b:0.32, SE:0.1, per unit, p ¼ 0.002) as well as with increased CIMT (b: 0.30, SE:0.12, per unit p ¼ 0.02), PWV (b: 0.201, SE:0.12, per unit p ¼ 0.04), male sex (b: 0.003, SE:0.001 p ¼ 0.016), and diabetes (b: 14, SE:7, p ¼ 0.047). No univariate association between medication and examined vascular markers or Lp-PLA2 was detected (p ¼ ns, data

Twelve patients out of 111 had an adverse cardiac event (4 had acute ST elevation MI, and 8 acute non-ST elevation MI) during 3 years of follow-up (34
6 months). Compared to patients without cardiac events, those with events had higher Lp-PLA2 (289
74 vs. 222
94, p ¼ 0.02), lower CFR (2.1
0.3 vs. 2.77
0.8, p ¼ 0.03) and RH-PAT (1.08
0.2 vs. 1.41
0.2, p ¼ 0.01) but similar IMT (1.45
0.4 vs. 1.25
0.6 mm,

Table 3 Correlations of vascular variables with Lp-PLA2 in a model of multivariate analysis. RHI-PAT

Model 1 Age Sex Hypertension Diabetes Dyslipidemiaa Smoking Mulivessel CAD Lp-PLA2 Model 2 Age Sex SBP LDLa FPG Smoking Mulivessel CAD Lp-PLA2

CFR

CIMT

PWV

beta

SE

p

beta

SE

p

beta

SE

p

beta

SE

p

0.077 8.694 7.837 2.333 9.990 1.360 0.076 0.48

0.3 8.6 8.6 8.4 8.2 7.2 0.09 0.1

0.845 0.323 0.260 0.780 0.240 0.850 0.444 1.2 mm, and PWV  10 m/ s were not related with outcome. (Log-rank chi-square: 0.08, p ¼ 0.8, and 0.07, p ¼ 0.8, respectively) (Fig. 1aee). By Cox regression analysis, Lp-PLA2 > 234.5 ng/dl, CFR < 2.54 and RH-PAT < 1.26 were independently associated with adverse cardiovascular events (HR: 5.0, 95%CI: 1.1e24.3, p ¼ 0.03, HR: 4.1, 95%CI:1.01e15.5, p ¼ 0.04 and HR: 3.9, 95%CI:1.0e16.3, p ¼ 0.047, respectively), in a multivariate model including CIMT > 1.2 mm (HR: 2.0, 95%CI: 0.7e 6.1, p ¼ 0.20) PWV  10 m/s (HR: 0.9, 95%CI: 0.3e2.8, p ¼ 0.81) as

well as age, sex, traditional cardiovascular risk factors, (diabetes, hypertension, smoking and dyslipidemia), presence of carotid plaques and multivessel CAD. 4. Discussion In our study cohort of patients with CAD, the increased concentration of Lp-PLA2 was associated with endothelial dysfunction as assessed by RHI-PAT, impaired CFR, and abnormal arterial wall properties as assessed by increased CIMT and PWV. The above vascular markers have an independent prognostic value for adverse cardiac events in CAD. Most importantly, this is the first study demonstrating Lp-PLA2 blood levels as an independent predictor of

Fig. 1. KaplaneMeier survival curve analysis showed that lipoprotein phospholipase A2 (Lp-PLA2) > 234.5 ng/dl, (a), coronary flow reserve (CFR) < 2.54 (b), and reactive hyperemia by pulse amplitude tonometry (RH-PAT) < 1.26 (c) were associated with death and/or myocardial infarction during a 3 year follow-up while carotid intima-media thickness (CIMT) >1.2 mm (d), and pulse wave velocity (PWV) 10 m/s (e), were not related with outcome.

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41

surrogate markers of vascular function and atherosclerosis namely, RH-PAT, CFR, CIMT and in particular of PWV, after controlling for traditional risk factors, in CAD patients. Additionally, Lp-PLA2 blood levels, RH-PAT and impaired CFR were associated with adverse outcome in our study cohort during a 3 year follow-up. When oxidative modification of LDL takes place, oxidized LDL particles penetrate the vessel wall and initiate the atherogenic process. Lp-PLA2 has been shown to play an active role in the oxidation of LDL and it is highly upregulated in atherosclerotic plaques [3]. The oxidative process transforms phosphatidylcholine (PC) to oxidative modified PC. This molecule acts as a substrate for Lp-PLA2. Thus, the interaction between oxidative modified PC and Lp-PLA2, generates the oxidized fatty acids (OxFA) and lysophosphatidylcholine (Lyso-PC) [1,3]. Lyso-PC and OxFA exert several proinflammatory actions (upregulation of adhesion molecules, cytokine and CD40 ligand expression, promotion of endothelial cell dysfunction, stimulation of macrophage proliferation, chemoattraction of inflammatory cells, production of metalloproteinases) that lead to atherosclerotic plaque formation and alteration of artrial wall properties. It has been shown that Lp-PLA2 molecules are expressed in and around the necrotic core of advanced human atheroma [4]. As the atheromatic plaque grows, its concentration in Lp-PLA2 is increased. Kolodgie at al, have shown an excessive staining for Lp-PLA2 within vulnerable atherosclerotic plaques [5]. As the products of Lp-PLA2 activity possess an important proinflammatory action, this enzyme is closely linked to the key steps in the progression of atherosclerosis and atherothrombosis. Oxidative stress, as measured by urinary excretion of 8-epi-prostagladin F2a, was also shown to be positively correlated with LpPLA2 activity in patients with angiographically proven coronary artery disease [6]. Oxidative stress, is a major determinant of endothelial dysfunction and coronary flow reserve [26]. Reactive hyperemia peripheral arterial tonometry (RH-PAT) is a method to assess peripheral microvascular endothelial function and is linked to coronary microvascular endothelial dysfunction [10] and higher cardiovascular event rates [11]. A reduced RHI-PAT has been related with presence of vulnerable coronary plaques as assessed by intravascular ultrasound [27]. Patients with CAD with a greater impairment of reactive hyperemia, as assessed by PAT have an adverse cardiovascular prognosis as assessed by combined elevation of CRP and Lp-PLA2 [28]. In line with these findings, our study documents an independent association of endothelial dysfunction, assessed by RH-PAT, with Lp-PLA2 levels after controlling for traditional atherosclerotic risk factors in CAD. Adenosine-induced CFR is also thought to be at least partly endothelium dependent [15,26]. Impaired CFR constitutes a marker of coronary microcirculation dysfunction and reflects the impairment of the epicardial coronary artery flow in the presence of significant coronary stenosis [13]. The scaling values of decreasing CFR constitute a comprehensive indicator of cardiovascular risk even in the presence of critical epicardial coronary stenosis [13]. This finding suggests that for the same degree of coronary epicardial stenosis decreasing values of CFR may also reflect coronary microcirculatory dysfunction. As with RHI-PAT, studies reported the independent association of coronary microvascular dysfunction, as assessed by CFR, with plaque predisposition to rupture in patients with CAD [29]. In the present study, CFR and RHI-PAT were interrelated suggesting the association of peripheral to coronary endothelial dysfunction in agreement with previous studies [11]. Furthermore, both non-invasive markers endothelial function were independently related to increasing levels Lp-PLA2 in blood. In particular patients with Lp-PLA2 > 234.5 ng/ml had a three fold risk to present a CFR < 2.54 and an RHI < 1.26 suggesting evidence of a significant coronary endothelial dysfunction in CAD patients with elevated Lp-PLA2. We may speculate that the local coronary release

39

of lysophosphatidylcholine and oxidation products by of Lp-PLA2 [1,3], provides a plausible biochemical mechanism for the interplay between Lp-PLA2 activity, endothelial dysfunction and CAD progression [4e6] Both impaired CFR and reduced RHI-PAT have proven prognostic value in CAD patients [11,13,14] Regarding the well-established independent prognostic role of Lp-PLA2 [2,30], our findings could implicate an biochemical link between endothelial dysfunction and increased cardiac event rate in CAD patients. Future studies will verify this hypothesis and will highlight the pharmaceutical modification of Lp-PLA2 as a promising therapy of high-risk CAD patients with CAD [22]. Increased CIMT has been consistently associated with most cardiovascular risk factors, the atherosclerotic lesions in other arterial sites, as well as with the presence and severity of CAD [15e 17]. Although initial evidence suggested an association of CIMT with Lp-PLA2 in non-diabetic subjects [31], this association was not confirmed in larger scale trials in the primary prevention setting [32]. It was suggested that the association of CIMT with Lp-PLA2 may be secondary to the distribution of Lp-PLA2 on LDL and HDL in various types of dyslipidemias and/or diabetes [33]. However, LpPLA2 has been shown to play an active role in the oxidation of LDL, the key-step of atherosclerosis progression [1,2] and it is highly upregulated in atherosclerotic plaques [4e6]. As the atheromatic plaque grows, its concentration in Lp-PLA2 also increases, implicating a parallel progression of atheromatic plaque with Lp-PLa2 expression [1,5]. In our study of CAD patients, elevated Lp-PLA2, blood levels predicted an increased CIMT and a 2-fold risk for the presence of carotid plaques and multivessel CAD independently of classical cardiovascular risk factors such as hypertension, dyslipidemia, or LDL levels. In agreement with our findings, recent studies have shown a relationship of Lp-PLA2 with coronary and carotid atherosclerosis severity in patients with cardiovascular disease [34]. The association of increased PWV with the presence and prognosis of angiographic CAD has been extensively demonstrated [35,36]. Lp-PLA2 [1] is a specific marker of vascular inflammation. On the other hand increased PWV is associated with enhanced vascular inflammation and injury [17,37]. However, the association between Lp-PLA2 and PWV has not been clarified. In the present study we have shown for the first time that in increasing Lp-PLA2 levels are related with increasing PWV values. Moreover, in the present study, we have found a independent association of circulating Lp-PLA2 in addition to age, systolic blood pressure and LDL with increased PWV. Our findings suggest an additive and independent role of this inflammatory biomarker to traditional atherosclerotic risk factors resulting to arterial stiffening. We have previously shown [17] that low grade inflammation, as assessed by CRP and increased metalloproteinase production are linked to increased PWV in CAD patients. Thus, Lp-PLA2 activity may be one the primary triggers of the proinflammatory process contributing to loss of elastin and increased collagen deposition by enhanced metalloproteinase activity leading to accelerated arterial stiffening. Furthermore, the association of Lp-PLA2 with markers of vascular function remained significant after adjustment for multivessel CAD. This finding indicates that the effect of Lp-PLA2 on vascular wall is present in both patients with single and multivessel CAD disease and thus, is independent of any interrelation between markers of vascular function and the extent of coronary atherosclerosis. Additionally, of all measured markers, Lp-PLA2, RH-PAT and CFR were associated with adverse outcome in our study cohort during a 3 year follow-up. However, the small number of cardiac events during follow-up does not allow to draw firm conclusions on the relative predictive value for adverse outcome of the measured vascular markers and Lp-PLA2, in the present study.

40

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41

4.1. Study limitations The limitation of the present investigation was the crosssectional design which prevented us from inferring definite causeeeffect relationships. Additionally, nearly all patients were on statins and thus we could not assess possible interactions of this treatment the reported correlations. Although, we found a close independent association of Lp-PLA2 with established non-invasive vascular markers of CAD prognosis, we cannot draw firm conclusions that the association of Lp-PLA2 with cardiovascular outcomes is mediated through its effects on the pathophysiology reflected by the examined surrogate markers of atherosclerosis. Lp-PLA2 was related with adverse outcome in our study cohort. However, the limited number of cardiac events during follow-up does not permit to draw firm conclusions on the relative predictive value for adverse outcome of the measured vascular markers and Lp-PLA2, and produces wide estimates for risk associated with Lp-PLA2, particularly when including adjustment for other covariates. Finally, it is known that the association of circulating Lp-PLA2 mass and activity with atherosclerotic manifestations weakens after adjustment for various cardiovascular risk factors [30,32]. Therefore, the clinical utility of Lp-PLA2 concentrations for risk stratification remains to be clarified in larger scale trials investigating various cohorts of patients with cardiovascular disease. 5. Conclusions In the present study, circulating Lp-PLA2 presents an independent to traditional risk factors, association with endothelial and coronary microcirculatory dysfunction as well as with the coronary, extra-coronary atherosclerosis and arterial stiffening in CAD patients likely because its powerful inflammatory action within the vascular wall. More specifically Lp-PLA2 was an independent determinant of RH-PAT, CFR, CIMT and PWV in the presence of CAD and was associated with adverse outcome in our study cohort during 3 years of follow-up. These findings suggest that the prognostic role of Lp-PLA2 in CAD may be mediated through its detrimental effects on endothelial function and arterial wall properties. The association of Lp-PLA2 with established vascular markers of prognosis also suggests that this biomarker may offer further risk stratification and reclassify patients with overt cardiovascular disease to a higher risk level suggestive of the need of an intensified treatment. It has been proposed that Lp-PLA2 could serve as a potential diagnostic and therapeutic target in CAD patients, especially in those with heavy atherosclerotic burden [1,38,39]. However, further investigation is needed to prove whether pharmaceutical reduction of Lp-PLA2 levels would improve vascular markers of atherosclerosis and thus, would beneficially affect prognosis in patients with CAD [22]. Conflict of interest The authors declare that they have no conflict of interest to report. References [1] Ikonomidis I, Michalakeas CA, Lekakis J, Parissis J, Anastasiou-Nana M. The role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in cardiovascular disease. Rev Recent Clin Trials 2011;6(2):108e13. [2] Anderson JL. Lipoprotein-associated phospholipase A2: an independent predictor of coronary artery disease events in primary and secondary prevention. Am J Cardiol 2008;101:23Fe33F. [3] MacPhee CH, Moores KE, Boyd HF, et al. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor. Biochem J 1999;338(Pt 2):479e87.

[4] Häkkinen T, Luoma JS, Hiltunen MO, et al. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol 1999;19:2909e17. [5] 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;26:2523e9. [6] Kim JY, Hyun YJ, Jang Y, et al. Lipoprotein-associated phospholipase A2 activity is associated with coronary artery disease and markers of oxidative stress in a case-control study. Am J Clin Nutr 2008;88:630e7. [7] Kuvin JT, Patel AR, Sliney KA, et al. Assessment of peripheral vascular endothelial function with finger arterial pulse wave amplitude. Am Heart J 2003;146:168e74. [8] Lekakis J, Abraham P, Balbarini A, et al. Methods for evaluating endothelial function: a position statement from the European Society of Cardiology Working Group on Peripheral Circulation. Eur J Cardiovasc Prev Rehabil 2011;18(6):775e89. [9] Nohria A, Gerhard-Herman M, Creager MA, Hurley S, Mitra D, Ganz P. Role of nitric oxide in the regulation of digital pulse volume amplitude in humans. J Appl Physiol 2006;101:545e8. [10] Bonetti PO, Pumper GM, Higano ST, Holmes DR, Kuvin JT, Lerman A. Noninvasive identification of patients with early coronary atherosclerosis by assessment of digital reactive hyperemia. J Am Coll Cardiol 2004;44:2137e41. [11] Rubinshtein R, Kuvin JT, Soffler M, et al. Assessment of endothelial function by non-invasive peripheral arterial tonometry predicts late cardiovascular adverse events. Eur Heart J 2010;31:1142e8. [12] Ikonomidis I, Tzortzis S, Paraskevaidis I, et al. Association of abnormal coronary microcirculatory function with impaired response of longitudinal left ventricular function during adenosine stress echocardiography in untreated hypertensive patients. Eur Heart J Cardiovasc Imaging 2012;13:1030e4. [13] Cortigiani L, Rigo F, Gherardi S, Bovenzi F, Picano E, Sicari R. Implication of the continuous prognostic spectrum of Doppler echocardiographic derived coronary flow reserve on left anterior descending artery. Am J Cardiol 2010;105: 158e62. [14] Sicari R, Rigo F, Cortigiani L, Gherardi S, Galderisi M, Picano E. Additive prognostic value of coronary flow reserve in patients with chest pain syndrome and normal or near-normally coronary arteries. Am J Cardiol 2009;103:626e63. [15] Ikonomidis I, Lekakis J, Papadopoulos C, et al. Incremental value of pulse wave velocity in the determination of coronary microcirculatory dysfunction in never-treated patients with essential hypertension. Am J Hypertens 2008;21: 806e13. [16] Coskun U, Yildiz A, Esen OB, et al. Relationship between carotid intima-media thickness and coronary angiographic findings: a prospective study. Cardiovasc Ultrasound 2009;7:59. [17] Ikonomidis I, Kadoglou N, Tsiotra PC, et al. Arterial stiffness is associated with increased monocyte expression of adiponectin receptor mRNA and protein in patients with coronary artery disease. Am J Hypertens 2012;25:746e55. [18] Kobayashi K, Akishita M, Yu W, Hashimoto M, Ohni M, Toba K. Interrelationship between non-invasive measurements of atherosclerosis: flowmediated dilation of brachial artery, carotid intima-media thickness and pulse wave velocity. Atherosclerosis 2004;173:13e8. [19] Xiong Z, Zhu C, Zheng Z, et al. Relationship between arterial stiffness assessed by brachial-Ankle pulse wave velocity and coronary artery disease severity assessed by the SYNTAX score. J Atheroscler Thromb 2012;19(11):970e6. [20] Tello-Montoliu A, Moltó JM, López-Hernández N, et al. Common carotid artery intima-media thickness and intracranial pulsatility index in non-ST-elevation acute coronary syndromes. Cerebrovasc Dis 2007;24:338e42. [21] Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: a systematic review and meta-analysis. J Am Coll Cardiol 2010;55:1318e27. [22] White H, Held C, Stewart R, et al. Study design and rationale for the clinical outcomes of the STABILITY Trial (STabilization of Atherosclerotic plaque By Initiation of darapLadIb TherapY) comparing darapladib versus placebo in patients with coronary heart disease. Am Heart J 2010;160:655e61. [23] Task Force Members, Montalescot G, Sechtem U, et al., ESC Committee for Practice Guidelines. ESC guidelines on the management of stable coronary artery disease: the Task Force on the management of stable coronary artery disease of the European Society of Cardiology. Eur Heart J 2013;34:2949e 3003. [24] Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, Writing Group on the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction, ESC Committee for Practice Guidelines (CPG). Third universal definition of myocardial infarction. Eur Heart J 2012;33: 2551e67. [25] Lanman RB, Wolfert RL, Fleming JK, et al. Lipoprotein-associated phospholipase A2: review and recommendation of a clinical cut point for adults. Prev Cardiol 2006;9:138e43. [26] Ikonomidis I, Lekakis JP, Nikolaou M, et al. Inhibition of interleukin-1 by anakinra improves vascular and left ventricular function in patients with rheumatoid arthritis. Circulation 2008;117:2662e9. [27] Schoenenberger AW, Urbanek N, Bergner M, Toggweiler S, Resink TJ, Erne P. Associations of reactive hyperemia Index and intravascular ultrasoundassessed coronary plaque morphology in patients with coronary artery disease. Am J Cardiol 2012;109:1711e6.

I. Ikonomidis et al. / Atherosclerosis 234 (2014) 34e41 [28] Heffernan KS, Karas RH, Patvardhan EA, Jafri H, Kuvin JT. Peripheral arterial tonometry for risk stratification in men with coronary artery disease. Clin Cardiol 2010;33:94e8. [29] Dhawan SS, Corban MT, Nanjundappa RA, et al. Coronary microvascular dysfunction is associated with higher frequency of thin-cap fibroatheroma. Atherosclerosis 2012;223:384e8. [30] Kleber ME, Wolfert RL, De Moissl GD, et al. Lipoprotein associated phospholipase A2 concentration predicts total and cardiovascular mortality independently of established risk factors (The Ludwigshafen Risk and Cardiovascular Health Study). Clin Lab 2011;57:659e67. [31] Constantinides A, van Pelt LJ, van Leeuwen JJ, et al. Carotid intima media thickness is associated with plasma lipoprotein-associated phospholipase A2 mass in nondiabetic subjects but not in patients with type 2 diabetes. Eur J Clin Invest 2011;41:820e7. [32] Garg PK, McClelland RL, Jenny NS, et al. Association of lipoprotein-associated phospholipase A(2) and endothelial function in the Multi-Ethnic Study of Atherosclerosis (MESA). Vasc Med 2011;16:247e52. [33] Tellis CC, Tselepis AD. The role of lipoprotein-associated phospholipase A2 in atherosclerosis may depend on its lipoprotein carrier in plasma. Biochim Biophys Acta 2009;1791:327e38.

41

[34] Charniot JC, Khani-Bittar R, Albertini JP, et al. Interpretation of lipoproteinassociated phospholipase A2 levels is influenced by cardiac disease, comorbidities, extension of atherosclerosis and treatments. Int J Cardiol 2013 Sep 20;168(1):132e8. [35] Ikonomidis I, Stamatelopoulos K, Lekakis J, Vamvakou GD, Kremastinos DT. Inflammatory and non-invasive vascular markers: the multimarker approach for risk stratification in coronary artery disease. Atherosclerosis 2008;199:3e11. [36] Orlova IA, Nuraliev EY, Yarovaya EB, Ageev FT. Prognostic value of changes in arterial stiffness in men with coronary artery disease. Vasc Health Risk Manag 2010;6:1015e21. [37] Kadoglou NP, Papadakis I, Moulakakis KG, et al. Arterial stiffness and novel biomarkers in patients with abdominal aortic aneurysms. Regul Pept 2012;179:50e4. [38] Mockel M, Danne O, Muller R, et al. Development of an optimized biomarker strategy for early risk assessment of patients with acute coronary syndromes. Clin Chim Acta 2008;393:103e9. [39] Davidson MH, Corson MA, Alberts MJ, et al. Consensus panel recommendation for incorporating lipoprotein-associated phospholipase A2 testing into cardiovascular disease risk assessment guidelines. Am J Cardiol 2008;101(Suppl.):51Fe7F.