Association between high‐sensitivity C‐reactive protein, lipoprotein‐associated phospholipase A2 and carotid atherosclerosis: A cross‐sectional study

Abstract High‐sensitivity C‐reactive protein (hs‐CRP) and lipoprotein‐associated phospholipase A2 (Lp‐PLA2) have been reported to be independent predictors of atherosclerosis. However, whether the combination of these two markers can improve the prediction of atherosclerosis is unknown. This study aimed to evaluate the association between combining hs‐CRP and Lp‐PLA2 and predicting carotid atherosclerosis. A total of 1982 participants aged ≥40 years were included in this study. Hs‐CRP and Lp‐PLA2 were measured by a high‐sensitivity nephelometry assay and quantitative sandwich enzyme‐linked immunosorbent assay, respectively. Ultrasonography was performed on the bilateral carotid arteries to evaluate stenosis and plaques. Multivariable logistic regression models were used to analyse the association between the combination of the hs‐CRP and Lp‐PLA2 levels and carotid plaques and stenosis. A total of 1579 (79.7%) and 181 (9.1%) subjects had carotid plaques and carotid stenosis, respectively. The group with high hs‐CRP and Lp‐PLA2 levels had the highest prevalence of carotid plaques (90.6%) and stenosis (20.8%). A significant association was found between high hs‐CRP and Lp‐PLA2 levels and carotid stenosis (adjusted odds ratio [OR]: 2.39; 95% confidence interval [CI]: 1.13‐5.09), but this combination was not associated with carotid plaques (OR: 2.62, 95% CI: 0.93‐7.38). The results suggested that the combination of hs‐CRP and Lp‐PLA2 were better predictors than either protein alone with regard to carotid atherosclerosis.

The results suggested that the combination of hs-CRP and Lp-PLA2 were better predictors than either protein alone with regard to carotid atherosclerosis.

K E Y W O R D S
carotid atherosclerosis, combination, high-sensitivity C-reactive protein, lipoprotein-associated phospholipase A2

| INTRODUCTION
Carotid atherosclerosis is a primary cause of stroke and disability worldwide. 1,2 Inflammation is an important predictor of carotid atherosclerosis and plays a critical role in the formation and development of plaques and enhancement of the intima media thickness (IMT). 3 Many inflammatory biomarkers, including lipoprotein-associated phospholipase A2 (Lp-PLA2), fibrinogen, serum amyloid A (SAA) and interleukin-6 (IL- 6), have been approved by the United States Food and Drug Administration (FDA) as predictors of ischemic stroke; additionally, high-sensitivity C-reactive protein (hs-CRP) has been associated with carotid atherosclerosis. 4 Hs-CRP is secreted by the liver in response to IL-6 following a microbial trigger or tissue damage. Numerous studies have shown that elevated hs-CRP levels are significantly associated with an increased risk of carotid atherosclerosis. 5,6 Indeed, adding CRP to the Framingham risk score improved the cardiovascular risk prediction. 7 Nevertheless, a significant association of hs-CRP with atherosclerosis has not been demonstrated in all epidemiological studies. 8,9 Compared to hs-CRP, Lp-PLA2 has the advantage of being a more specific marker for cardiovascular risk. 7 However, epidemiological studies conducted in different populations have found inconsistent results regarding whether Lp-PLA2 can be used as a predictor of atherosclerosis. 10 This controversy has fuelled the search for more effective prediction methods for cardiovascular disease.
Several prospective studies have demonstrated that the combination of hs-CRP and Lp-PLA2 improves the prediction of the risk of cardiovascular diseases, including coronary artery disease and stroke. 7,11 However, the association of hs-CRP with Lp-PLA2 for carotid atherosclerosis is less understood. The aim of this study was to evaluate the association of the combination of hs-CRP and Lp-PLA2 with carotid atherosclerosis in participants with 40 years or older, who participated in the Asymptomatic Polyvascular Abnormalities Community (APAC) study. The carotid atherosclerosis risk prediction obtained by measuring hs-CRP and Lp-PLA2 was discussed.

| Study population
The APAC study is a community-based, observational cohort study aimed at investigating the epidemiology of asymptomatic polyvascular abnormalities in Chinese adults. 12,13 The inclusion crite- Written informed consent was obtained from all participants.

| Assessment of carotid stenosis and carotid plaque
Bilateral carotid duplex sonography (ACUSON X300, Siemens, Germany) was used to evaluate the presence of carotid stenosis and carotid plaques. 1, 12 The investigators were blinded to the clinical and laboratory examination results of the study participants. A carotid plaque was defined as a focal structure either invading >0.5 mm into the arterial lumen, or invading into at least 50% of the surrounding IMT, or with a thickness at least 1.5 mm from the intimae-lumen interface to the media adventitia interface. Carotid stenosis was assessed using established ultrasound criteria as follows 1 : (a) an internal carotid artery peak systolic velocity ≤125 cm/s was defined as <50% stenosis; (b) an internal carotid artery peak systolic velocity 125-230 cm/s and a visible plaque was defined as 50%-69% stenosis and (c) an internal carotid artery peak systolic velocity >230 cm/s or a markedly narrowed lumen was defined as >70% stenosis. The carotid ultrasound examination results were reviewed by two independent reviewers. Discrepancies were resolved by discussion between the experts or a third examination.

| Covariates
Questionnaires were used to collect information from the participants, including demographic variables, history of disease, lifestyle and drug history. Clinical characteristics and biochemical indicators were assessed at Kailuan General Hospital. The covariates included the education level, monthly per capita income, smoking, alcohol intake, blood pressure, fasting blood glucose, body mass index (BMI), cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). The assessment of covariates was described in a previous study. 2

| Statistical analysis
All continuous variables in our study were normally distributed. Continuous variables were presented as the mean with standard deviation and compared using one-way ANOVA. Categorical variables were compared using the chi-square test. Logistic regression was used to evaluate the association between hs-CRP, Lp-PLA2 and car- All statistical tests were two-sided, and the significance level was set as P < 0.05. The statistical analyses were performed using SAS software, version 9.3 (SAS Institute Inc., Cary, NC, USA).

| Participant characteristics
Of the 5440 participants in the APAC study, 5349 and 2012 participants had recorded measurements for hs-CRP and Lp-PLA2, respectively. The prevalence of carotid plaques and carotid stenosis were 79.7% (1579/1982) and 9.1% (181/1982), respectively. The characteristics of the participants are summarized in Table 1. Significant differences were found among the groups in age, education level, income, BMI, smoking status, alcohol consumption, hypertension, diabetes, total cholesterol, triglycerides and HDL-C (P < 0.05). The prevalence of carotid plaques and carotid artery stenosis was higher in the hs-CRP+/Lp-PLA2+ group (90.6% and 20.8%, respectively, all P < 0.05) than in the other three groups (Figure 2).

| Association between hs-CRP, Lp-PLA2 and carotid plaques and carotid stenosis
The associations between hs-CRP combined with Lp-PLA2 for carotid plaques and carotid stenosis are shown in Figure 3

| DISCUSSION
In this community-based study, the main finding was that the combi-  concentrations are significantly associated with atherosclerosis characterized by artery plaques and artery stenosis. 18,19 Significant associations have also been found between CRP combined with Lp-PLA2 and atherosclerotic events, such as coronary heart disease and stroke. 7,20 An elevated Lp-PLA2 level was shown to be significantly associated with carotid artery plaques, but this association became non-significant when Mendelian randomization methods were adopted. 21 Our findings provide evidence that the combination of the hs-CRP and Lp-PLA2 levels is sufficiently stable to predict carotid artery stenosis independent of other atherosclerotic risk factors. The significant association between high hs-CRP and Lp-PLA2 levels and carotid artery stenosis was still present even after the multivariable adjustments, demonstrating that hs-CRP combined with Lp-PLA2 is an independent risk factor for carotid artery stenosis. The elevated  In conclusion, our results indicated that the combination of CRP and Lp-PLA2 was associated with carotid stenosis. Our findings suggested that the combination of the CRP and Lp-PLA2 levels might be a useful measure for vascular risk assessment and could be a potential therapeutic target for carotid atherosclerosis.

ACKNOWLEDG EMENTS
We are grateful to the Kailuan Group for its collaboration, especially the dedicated participants, and all of the research staff involved in the study.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest. F I G U R E 3 Association of hs-CRP combined with Lp-PLA2 and carotid plaques and carotid stenosis. *hs-CRP+, high hs-CRP; hs-CRP−, low hs-CRP; Lp-PLA2−, low Lp-PLA2; Lp-PLA2+, high Lp-PLA2; High hs-CRP was defined as a CRP concentration ≥3 mg/L, High Lp-PLA2 was defined as a Lp-PLA2 concentration ≥200 ng/mL; Model 1: unadjusted; Model 2: adjusted for age, sex, education, income, smoking, and alcohol consumption; Model 3: adjusted for model 2 plus hypertension, hyperlipidemia, diabetes mellitus, and BMI