Association between lipid profiles and presence of carotid plaque

It is indicated that lipids profiles are associated with carotid plaque and Atherosclerosis. However, studies about the relationship between serum lipid profiles and carotid plaque composition in Chinese Population is limited. We conducted a cross-sectional study among 3,214 participants between January 2015 and December 2017 in China, to investigate the association between various lipid profiles and the prevalence of carotid plaque. Logistic regression model was used to investigate the association between plasma lipid profiles and odds of carotid plaque. Analysis of covariance (ANCOVA) was used to compare the mean plasma lipid profiles among different number and composition of carotid artery plaques. HDL-C, Non-HDL-C levels, TC/HDL-C, LDL-C/HDL-C were significantly associated with the presence of carotid plaque; HDL-C, LDL-C, Non-HDL-C levels, TC/HDL-C, LDL-C/HDL-C were significantly associated with the presence of common carotid artery (CCA) plaque. Compare with participants without carotid plaque, increased level of LDL-C/HDL-C was found in those with echolucent/polytype plaque. Similarly, compared with participants without CCA plaque, increased level of LDL-C/HDL-C was found in those with echolucent plaque. In conclusion, we found that serum HDL-C, Non-HDLc level, TC/HDLc, and LDLc/HDLc were all associated with the prevalence of carotid plaque, and LDL-C/HDL-C differed among different group of carotid plaque composition.


Discussion
To the best of our knowledge, this is the largest study in China to investigate the association between lipid profiles and the prevalence of carotid plaque and carotid plaque composition. The main results are as follows: serum HDL-C level was negatively associated with odds of carotid plaque and CCA plaque; serum LDL-C level was positively associated with odds of CCA plaque; serum Non-HDLc level, TC/HDLc, and LDLc/HDLc were positively associated with odds of both carotid plaque and CCA plaque. LDL-C/HDL-C differed among different group of carotid plaque composition.
Advanced atherosclerotic plaques are characterized as lipid nucleus which were covered with fibrous caps consisting of extracellular matrix and smooth muscle cells 18 . Several procedure associated with the progression of the atherosclerotic lesions(e.g. lipids accumulation, thrombosis, proteolysis, inflammation, and apoptosis) might be relevant with the development of plaque. It has been demonstrated that carotid plaque, especially the thick and irregular ones were risks factor for cardiovascular disease [19][20][21] . Therefore, identification of serum biomarkers related to the presence of plaque would be of great advantage in the prevention of atherosclerosis and CVD. The various lipid profiles examined in our study were related with carotid plaque, thus might be correlated with CVD. Previous studies suggested that lipid profiles have pivotal roles in the pathophysiology of carotid plaque [10][11][12][13][14][15][16][17] , which is consistent with our study. In contrast, one study in USA found no significant association between HDL-C or TG and carotid plaque, but the authors reported that higher TC, LDL, or non-HDL was associated with higher risk of carotid plaque 22 . An Algeria cross-sectional study also suggested inconsistent findings: lower HDL-C is associated with higher prevalence level of carotid plaque, but not LDL-C 23 . The inconsistency might be ascribed to many factors, including the study population heterogeneity, various study regions and different study designs.
Ultrasound (US), a non-radiative and cost-effective technique, is a patient-friendly an ideal method for immediate diagnosis and follow-up 24 . Previous studies have demonstrated that hypoechoic plaques (echolucent) are more often associated with cardiovascular symptoms than hyperechoic ones (echorich). The meta-analysis conducted by Jashari et al. demonstrated that carotid plaque echogenicity was associated with increased risk of CV events 25 . However, evidence regarding the relationship between serum lipid profile and carotid plaque composition in Chinese Population is limited. Our study found that LDL-C/HDL-C level was increased in those with echolucent plaque than those without carotid plaque. The analysis from our study provides new evidence for the associations between plaque characterization/features and health related outcomes.
The potential pathophysiological mechanisms through which lipid profiles might increase the risk of carotid plaque are not fully understood. First, the main beneficial effect of HDL cholesterol is to reverse the transport of cholesterol, remove free cholesterol from endothelial macrophages, and return it to the liver for excretion into bile, thus preventing the formation of atherosclerotic plaques 26  www.nature.com/scientificreports www.nature.com/scientificreports/ has both direct and indirect anti-inflammatory effects with potential antithrombotic results 27 .Third, the LDL-C/ HDL-C ratio is closely correlated with the distribution of HDL-C subclasses. With the increase of LDL-C/HDL-C ratio, the size of HDL-C particles generally decreases, suggesting that the maturation process of high density lipoprotein is blocked, which may be one of the contributors for the progress of atherosclerosis 28 .
There were some strengths in this study. First, we collected information on several potential confounders, and the potential effects of them were taken into account. Second, we firstly investigated the association of various lipid parameters and carotid plaque composition in Chinese Population. Third, subgroup analyses generally yielded similar results, indicating that the associations are robust.
Despite these strengths, the study also had several limitations. First, this is a cross-sectional analysis, thus it is difficult to detect the temporal relationship. Second, our study was carried out in a single-center, and these finding should be verified in multi-center study with larger populations. Last, we used ultrasound to assess the presence of plaque, which may be less reliable than the high-resolution magnetic resonance imaging (MRI) or computed tomography (CT). However, one study investigating the accuracy of ultrasound, CT, and MRI in diagnosis of carotid plaque morphology found that ultrasound has higher accuracy for diagnostics of carotid plaque morphology than CT or MRI 29 . In addition, compared with CT or MRI, ultrasound is safe, noninvasive, and inexpensive 30 . www.nature.com/scientificreports www.nature.com/scientificreports/ In summary, our study demonstrated that serum HDL-C level was associated with lower odds of carotid plaque and CCA plaque; serum LDL-C level was associated with higher odds of CCA plaque; serum non-HDL-C level, TC/HDL-C ratio, and LDL-C/HDL-C ratio were associated with higher odds of carotid plaque and CCA plaque. LDL-C/HDL-C differed among different group of carotid plaque composition. Further prospective studies with larger sample size are needed to verify these associations.

Methods
Study population. During January 2015 and December 2017, 3,214 consecutive participants (2,212 men, 1,002 women) who underwent a standard medical screening at the Physical Center of the First Affiliated Hospital of Zhengzhou University, China, were included. We excluded participants with the following conditions that might seriously influence carotid plaque and lipid profiles: (1) aged less than 18 years old; (2) with a history of cardiovascular disease, peripheral arterial disease, oncology disease, infectious disease, or serious liver or renal disease; (3) without data on lipid profiles or carotid ultrasound measurements; (4) taking medicine when undergoing the medical examination.

Lipid profiles assessment.
Overnight fast blood samples were collected in the morning. Plasma levels of TC, triglycerides (TG), HDL-C, and LDL-C were measured by a fully automatic analyzer, ROCHE (Roche Molecular Systems, Inc. Basel, Switzerland) module Cobas 8000 (C701/C702/C502), and kits were procured by ROCHE. Quality control is maintained by the laboratory with standard procedures. The coefficient of variation for repeated measurements on samples from individual hospitalized patients is maintained at ≤2.5%.
Carotid ultrasound assessment. Carotid ultrasound was assessed by B-mode ultrasound with an Aplio 300 ultrasound system (Toshiba Medical Systems, Tokyo, Japan) by trained and certified technologists. With the subject lying in a supine position, the extracranial carotid arteries were imaged in the longitudinal (anterior, lateral, and posterior views) and transverse planes, the common carotid artery (CCA), the internal carotid artery (ICA), and the external carotid artery (ECA) on both sides were carefully scanned under B-mode imaging from multiple directions. The IMT was defined as the distance between the leading edge of the lumen-intima echo and the leading edge of the media adventitia echo. The carotid intima-media thickness (CIMT) was defined as the mean of the right and left IMT of the CCA.
Carotid plaque is defined as the presence of focal wall thickening that is at least 0.5 mm, or 50% greater than that of the surrounding vessel wall, or as a focal region with CIMT greater than 1.5 mm that protrudes into the lumen that is distinct from the adjacent boundary 31 . Plaques were classified as echolucent (low grayscale  www.nature.com/scientificreports www.nature.com/scientificreports/ median, lipid rich), echogenic (high grayscale median, mostly occupied by calcified areas), and heterogeneous (mixed echolucent and echogenic) 32 . If all the plaques had the same composition, the participant was classified as plaque composition, those who had different composition were defined as polytype plaques. All carotid artery plaque measurement was conducted by the same trained physician using the same B-mode ultrasound. The study adopted strict quality control procedures for monitoring and testing consistency in image acquisition and image analysis.

Covariates.
A standard physician-administered questionnaire was used to collected information on the demographic characteristics, lifestyle factors, and medical history by trained physicians. Participants who smoked at least one cigarette per day or drank alcohol once a week for at least six months were defined as smokers or drinkers. Weight and height were measured on an auto-anthropometer with subjects wearing light clothing and no shoes. Body mass index (BMI) was calculated as the weight (kg) divided by the square of the height (m). Blood pressure was measured using an electronic device by trained physicians after the subjects sitting relaxed for 10 mins.
Statistical analyses. Descriptive data are presented as mean (standard deviation) for continuous variables and number (percentage) for categorical variables.
Logistic regression model was used to examine the association between plasma lipid profiles and odds of carotid plaque. And two models were developed: Model 1: adjusted for age (years, continuous variable), sex (male or female), and BMI (continuous variable, kg/m 2 ); Model 2: further adjusted for systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting plasma glucose, uric acid, TC (not for TC vs. carotid plaque), TG (not for TG vs. carotid plaque), HDL-C (not for HDL-C vs. carotid plaque) and LDL-C (not for LDL-C vs. carotid plaque).
Analysis of covariance (ANCOVA) was used to compare the mean plasma lipid profiles among different number and composition of carotid artery plaques. The two aforementioned models were also adopted. Subgroup analyses were conducted to investigate whether the relationships between plasma lipid profiles and carotid plaque were modified by sex (men or women) or BMI (<25 kg/m 2 or ≥25 kg/m 2 ). Each potential modifier was examined in a separate model by adding a multiplicative interaction term (i.e. potential modifier * lipid profiles).
All analyses were performed with SPSS 21.0 for Windows (SPSS, Inc., Chicago, USA). Two-sided bonferroni correction P values < 0.01 for the difference of covariate-adjusted mean (SEM) among different composition/ number of carotid plaque (i.e. None, Echolucent, Echogenic, Heterogeneous, and Polytype). Two-side P values < 0.05 were considered statistically significant for the other associations.  Table 4. Covariate-adjusted mean (SEM) lipid profiles according to different composition of common carotid artery plaque. Compared with participants without CCA plaque, *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001.