Association of lipoprotein‐associated phospholipase A2 mass with asymptomatic cerebral artery stenosis

Abstract Cerebral artery stenosis (CAS) is the most important causes of ischaemic stroke. Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) plays 2 diverse roles in atherosclerosis (pro‐inflammatory and anti‐inflammatory), and the association between Lp‐PLA2 mass and cardiovascular or cerebrovascular events is inconsistent among previous studies. A cross‐sectional study including 2012 North Chinese adults aged ≥40 years was performed in 2010‐2011 to investigate whether Lp‐PLA2 mass is associated with asymptomatic cerebral artery stenosis (ACAS). Serum Lp‐PLA2 mass was determined by enzyme‐linked immunosorbent assay (ELISA). All participants underwent transcranial Doppler (TCD) and bilateral carotid duplex ultrasound to evaluate intracranial artery stenosis (ICAS) and extracranial arterial stenosis (ECAS). The median serum Lp‐PLA2 mass of the participants was 140.74 ng/mL (interquartile range: 131.79‐158.07 ng/mL). The adjusted odds ratio (OR) when comparing the 4th quartile to the 1st quartile of Lp‐PLA2 was 1.98 (95% confidence interval (CI): 1.42‐2.78), 1.79 (95% CI: 1.08‐2.94) and 1.87 (95% CI: 1.28‐2.73) for the occurrence of ACAS, asymptomatic ECAS and asymptomatic ICAS, respectively, after controlling for vascular risk factors. These independently significant associations remained statistically significant in the male or elderly subgroups, but not in females or middle‐aged participants. Lp‐PLA2 mass is positively correlated with subclinical atherosclerosis determined by ACAS, ICAS and ECAS in North Chinese, particularly in male and older participants, suggesting that serum Lp‐PLA2 mass might be potential biomarker for the detection of ACAS in the adults.

after controlling for vascular risk factors. These independently significant associations remained statistically significant in the male or elderly subgroups, but not in females or middle-aged participants. Lp-PLA2 mass is positively correlated with subclinical atherosclerosis determined by ACAS, ICAS and ECAS in North Chinese, particularly in male and older participants, suggesting that serum Lp-PLA2 mass might be potential biomarker for the detection of ACAS in the adults. for approximately 20% of all causes of death. 2 The prevalence of intracranial atherosclerotic disease in China is higher than that in other populations, being responsible of as far as one-third of strokes. 3 Currently, the diagnosis of ischaemic stroke (IS) is mainly based on the experience of clinicians and brain imaging results.
However, many patients with a suspected stroke are not assessed in a timely manner (within the first few hours after the event). Thus, establishing an accurate and quick screen procedure in patients with suspected acute ischaemic stroke is very important. 4 Identification of serum biomarkers is one of the primary strategies used to identify populations at risk of ischaemic stroke. Among these strategies, lipoprotein-associated phospholipase A2 (Lp-PLA2) has been identified as a promising soluble blood-based biomarker. 5 Lp-PLA2 is a type of serine lipase, majority of which bound to lowdensity lipoproteins (LDL) and less of which bound to high-density lipoprotein (HDL) or very low-density lipoprotein (VLDL). 6 Paradoxically, the role of Lp-PLA2 in the progression of atherosclerosis is controversial. [7][8][9] As a well-established pro-inflammatory factor, Lp-PLA2 is extensively involved in the progression of atherosclerosis, including plaque formation, development and rupture. 10 A high level of Lp-PLA2 has been proved to be a positive risk factor for cardiovascular and cerebrovascular events in multiple large-scale population studies. 4 11 In contrast, some studies demonstrated a decrease in Lp-PLA2 enzymatic activity in patients with acute myocardial infarction and no correlation with premature coronary atherosclerosis; 16,17 therefore, the underlying mechanism remains unclear.
Lp-PLA2 is well recognized as a potential biomarker for the vascular inflammation and formation of rupture-prone plaques. 18 Lp-PLA2 is considered to predict the risk of not only first-ever but also recurrent strokes, [19][20][21][22] but it does not correlate with acute cerebrovascular ischaemic diseases. 23 This discrepancy might be due to the heterozygous phenotypes of stroke and the paradoxical roles of Lp-PLA2 in the inflammation. Lp-PLA2 mass is significantly associated with isolated ICAS and concurrent extra-intracranial stenosis but not related to isolated ECAS, in a stroke-free hypertension population from North China. 24 In this study, we examined asymptomatic cerebral artery stenosis (ACAS), an intermediate indicator that can remain silent for a long period before the occurrence of ischaemic stroke, 25 and suggested that aberrant increase in Lp-PLA2 mass might be associated with ACAS in a vascular disease-free population. The objective of this study was to investigate the association between serum levels of Lp-PLA2 with ACAS in a Chinese cohort, with the aim of identifying useful and novel biomarkers for the prediction of stroke at an early stage.

| Study design and population
The participants and design of the Asymptomatic Polyvascular Abnormalities in Community (APAC) study can be referred to your previously description. 26 The APAC study included a random sample of 5440 participants aged ≥40 years. The participants were selected from the baseline population from the Kailuan Study and included 101 510 current employees and retirees from the Kailuan (Group) Co. Ltd. 26 The APAC study protocol has been previously reported in detail. 26 Among the 5440 participants, 2012 with complete demographic and blood sample information were randomly selected to investigate the association between Lp-PLA2 mass and ACAS.

| Measurement of Lp-PLA2
The correlation between Lp-PLA2 mass and Lp-PLA2 activity was about 0.50. 11 In addition, the Lp-PLA2 mass and Lp-PLA2 activity had similar predictive power for the cardiac death 27 and stroke. 28 Therefore, we measured Lp-PLA2 mass only.
Venous bloods were collected in the fasting condition, and EDTA was used as an anticoagulant. The blood samples were centrifuged for 5 minutes at 500 9 g. within 2 hours of field collection, and serum was separated and placed in microcentrifuge tubes. Blood samples had unified number for each participant and were stored at À80°C. To reduce the interassay error and measurement error, the Lp-PLA2 mass for all participants was assessed simultaneously by a professional technician using the human Lp-PLA2 enzyme immunoassay kit (CUSABIO, Wuhan, China) at Beijing Tiantan Hospital, Capital Medical University, Beijing, China, according to the manufacturer's instructions.

| Assessment of cerebral artery stenosis
Intracranial artery stenosis (ICAS) was assessed through the use of a transcranial Doppler (TCD) by 2 independent experienced neurologists using portable machines (EME, Companion, Nicolet), according to standardized protocol and diagnosis criteria. 26 Artery stenosis was defined by the peak systolic flow velocity as follows: >140 cm per second for the middle cerebral artery, or >120 cm per second for the anterior cerebral artery, or >100 cm per second for the posterior cerebral artery and vertebral-basilar artery or >120 cm per second for the ICAS. Except for velocity criteria, the presence of turbulence or background noise, and whether the abnormal velocity was considered to be segmental.
Every participant also underwent a bilateral carotid duplex ultrasound (Philips iU-22 ultrasound system, Philips Medical Systems, Bothell, WA, USA) to assess extracranial arterial stenosis (ECAS).
Bilateral ECAS arteries included common carotid arteries, carotid bifurcation, the internal carotid artery and the external carotid artery.
All participants were examined in the supine position with the head turned to the contralateral side. Both sides of the carotid arteries were evaluated for the presence of ECAS (≥50%), which was graded based on recommendations from the Society of Radiologists in Ultrasound Consensus Conference. 29 ACAS was defined by the presence of at least one of ECAS or ICAS.

| Covariates
Demographic variables including age, sex and history of hypertension, diabetes mellitus and dyslipidaemia were collected via questionnaires. All participants were divided into 2 groups based on their ages: 40-59 years and ≥60 years. Information regarding disease history, including hypertension, diabetes mellitus or hyperlipidaemia, as well as smoking history, which was classified as "yes" or "no" was also collected via questionnaires. Weights (accurate to 0.1 kg) and heights (accurate to 0.1 cm) were measured during the physical examination, and body mass index (BMI) values were calculated.
Hypertension was defined as the presence of hypertension history, use of antihypertensive medication, a systolic blood pressure (SBP) ≥140 mm Hg or a diastolic blood pressure (DBP) ≥90 mm Hg. Diabetes mellitus was defined by self-reported history, current use of insulin or oral hypoglycaemic agents, or a fasting blood glucose level ≥7.0 mmol/L (126 mg/dL). Dyslipidaemia was defined as a selfreported history, current use of cholesterol-lowering medication, total cholesterol (TC) level ≥220 mg/dL or triglycerides (TG) ≥150 mg/dL or low-density lipoprotein cholesterol (LDL-C) ≥160 mg/dL. C-Reactive protein (CRP) was measured by high-sensitivity nephelometry assay (Cias Latex CRP-H; Kanto Chemical Co. Inc, Tokyo, Japan). CRP concentrations were categorized into 2 groups according to the guideline. 30 All blood examinations were performed at the central laboratory of Kailuan Hospital.

| Statistical analyses
Continuous variables are presented as the mean AE standard deviation or median together with interquartile range, whereas categorical variables are presented number together with percentages. Continuous variables were compared using analysis of variance (ANOVA) for normally distributed variables and nonparametric approach for skewed distributed variables. The intergroup differences in categorical variables were compared using chi-square tests. We used logistic regression to determine the association between the Lp-PLA2 mass and cerebral artery stenosis risk and represented as odds ratio (OR) and 95% confidence interval (CI). Known confounding factors including age, sex, BMI, current smoker, alcohol use, hypertension, diabetes mellitus and dyslipidaemia were controlled in the logistic regression. All statistical tests were 2-sided, and P < .05 was considered to be statistically significant. All analyses were performed with SAS (version 9.1; SAS Institute, Cary, NC, USA) software.

| Ethics statement
This study was approved by the Ethics Committees of Beijing Tiantan Hospital, Capital Medical University. All participants signed informed consent forms before the participation in this study.  The association between artery stenosis and Lp-PLA2 mass was summarized in Table 2

| DISCUSSION
The present study indicated that the serum Lp-PLA2 mass was elevated with ACAS. The results demonstrated that elevated Lp-PLA2 mass is independently associated with the increased risk of ACAS (either ICAS or ECAS), and the independent association is statistically significant in the male or elderly subgroups, but not significant in females or middle-aged participants. To our knowledge, this is the first attempt to investigate the association between Lp-PLA2 mass and the presence of subclinical atherosclerosis in a general and asymptomatic Chinese population, and the only study addressing the association between Lp-PLA2 mass and ACAS, ICAS or ECAS in terms of general population levels.
We found that a high Lp-PLA2 mass (eg 4th quartile) was associated with an increased risk of ACAS. Our findings were in part consistent with the findings reported in another population (stroke-free hypertensive patients), which showed that Lp-PLA2 mass was inde- Recently, a systematic review was performed to explore the associa- The present study revealed that the associations between Lp-PLA2 mass and ACAS were statistically significant in males but not in females. This finding is consistent with the results of the Dallas Heart Study, which has shown that Lp-PLA2 mass is modestly associated with CAC in males but not in females. 32 Our finding is similar to the results of the Chinese Multiprovincial Cohort Study-Beijing Project, which showed that Lp-PLA2 activity is independently associated with the development of subclinical atherosclerosis in men but not in women. 33 Oestrogen has been reported to be associated with the low expression or activity of Lp-PLA2; 34  In the present study, the association between ICAS or ECAS was significant only in elderly participants (aged ≥60 years) but not in middle-aged participants (<60 years). This finding is consistent with results from a report investigating a Japanese population, 37

| CONCLUSION
In conclusion, we found that Lp-PLA2 mass was positively correlated with subclinical atherosclerosis determined by ACAS, ICAS and ECAS, particularly in male and older participants. Considering that elevation Lp-PLA2 contributes about 2-fold risk for strokes or coronary artery disease, testing for Lp-PLA2 might be a supplementary evaluation tool to classical cardiovascular risk assessment.

ACKNOWLEDG EMENTS
This work was supported by grants from the National Natural

CONFLI CT OF INTEREST
The authors declare that they have no conflict of interest.