Correlation between plasma levels of Lp-PLA2, Hcy, CRP, Lp(a), AT-III, and TEG parameters and carotid atherosclerosis in patients with combined hypertension and cerebral infarction

Background: To observe the correlation between Lp-PLA2, Hcy, CRP, Lp(a), AT-III, and TEG parameters and carotid atherosclerosis with combined hypertension and cerebral infarction and evaluate their value in risk determination. Methods: Patients with primary hypertension were selected as subjects and divided into 2 groups based on cerebral infarction: simple hypertension group and hypertension combined with cerebral infarction group. The differences of Lp-PLA2, Hcy, CRP, Lp(a), AT-Ⅲ, and TEG were compared. Spearson correlation and multivariate logistic regression model were used to analyze the correlation. A ROC curve was used to analyse the value of a single item and their combination for the determination of carotid AS risk. Results: The levels of single item and CIMT in the hypertension combined with cerebral infarction group were higher, and the values of R, EPL, and LY30 were lower than corresponding indicators in the simple hypertension group. Furthermore, it was found that Lp-PLA2 and Hcy were risk factors. The AUC for Hcy and Lp-PLA2 for the determination the carotid AS risk were larger. Conclusions: The increase or decrease of Lp-PLA2, Hcy, and TEG were important factors influencing the development of carotid AS with combined hypertension and cerebral infarction. The levels of Lp-PLA2, Hcy, and TEG with combined hypertension and cerebral infarction were significantly different from those with simple hypertension and could be used as independent predictive factors for determining carotid AS risk.

markers is very important for predicting the development of cerebral infarction, evaluating the disease condition of patients, and scientifically selecting treatment regimens. Some recent studies have shown that several mediators such as lipoprotein-associated phospholipase A2 (Lp-PLA2), homocysteine (Hcy), C-reactive protein (CRP), and lipoprotein(a) (Lp(a)) might be risk factors for AS (17)(18)(19).
Carotid intima thickening is an early sign of AS, therefore, it is selected as a predictive indicator of cardiovascular and cerebrovascular events. Carotid intimamedia thickness (CIMT) is detected by colour Doppler ultrasound as the standard, to screen for plasma inflammation and coagulation-related indicators (Lp-PLA2, Hcy, CRP, Lp(a), AT-Ⅲ, and TEG) that participate in AS plaques and thrombosis to investigate the correlation between each indicator and CIMT in order to provide bases for the prevention of AS and treatment of patients with combined hypertension and cerebral infarction. blood pressure at admission indicated stage 2-3 hypertension; systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥100 mmHg; no previous history of peripheral arteriosclerosis and cardiovascular and cerebrovascular diseases; and no positive sign during the physical examination; ② patients who met the diagnostic criteria of cerebral infarction revised during the Fourth National Cerebrovascular Disease Conference and in whom the diagnosis was confirmed by magnetic resonance imaging (MRI) or brain computed tomography (CT); and ③ healthy individuals who received a physical examination, did not have previous history of peripheral arteriosclerosis and cardiovascular and cerebrovascular diseases after inquiry of the medical history, did not have diseases that caused increased levels of Lp-PLA2, Hcy, CRP, and Lp(a), and had not recently taken any medication. The exclusion criteria were ① patients who had secondary hypertension, atrial fibrillation, diabetes mellitus, coronary heart disease, and malignant tumours; ② patients who had kidney and coagulation diseases and thyroid dysfunction; ③ patients who had haematopoietic diseases, rheumatic and immune system diseases, tissue necrosis, and acute and chronic inflammation diseases combined with infection and fever; ④ patients with a history of surgery, trauma, deep vein thrombosis, and organ transplantation; ⑤ patient who took medication that affected Lp-PLA2, Hcy, CRP, and Lp(a) levels (such as diuretics, folic acid, vitamin B complex, and statins); ⑥ patients with a history of long-term smoking and drinking; and ⑦ pregnant or lactating women. The simple hypertension group had 193 patients (106 males and 87 females) whose ages ranged from 23 to 91 years (average age, 64±14 years). The hypertension combined with cerebral infarction group had 192 patients (117 males and 75 females) whose ages ranged from 34 to 90 years (average age, 69±12 years). In addition, a healthy population who received a physical examination was used as the control group. In the control group, there were 151 patients (105 males and 46 females), whose ages ranged from 31 to 85 years (average age, 65±13 years). These people of control group had never had a stroke or a history of high blood pressure. Model 680 enzyme-labeled instrument (Bio-Rad, America) with a functional assay sensitivity of 10 ng/ml. The levels of Hcy were measured by an ARCHITECT i2000SR immunoanalyzer (Abbott, America) with a functional assay sensitivity of 1.0μmol/L. The CRP levels were detected by a QuikRead go immunity analyzer (Orion Diagnostica Oy, Finland) with a functional assay sensitivity of 1.0 mg/L. The levels of Lp(a) were measured by an OLYMPUS AU5800 biochemical analyzer (Beckman Coulter, America). The levels of AT-Ⅲ were measured by a STA-R Evolution coagulation analyzer (STAGO, France). Simulation of the coagulation process in the body and dynamic monitoring of coagulation system functioning are used to detect various indicators such as the coagulation factor activity, platelet function, and fibrinolytic activity. The detection parameters in this study included coagulation reaction time (R), clot formation time (K), clot formation rate (α-angle), maximum clot strength or hardness (MA), estimated percent lysis (EPL), clot amplitude reduction rate (LY30), coagulation comprehensive index (CI), clot mechanic strength (G), and platelet aggregation function (A). We detected TEG with a Thrombelastograph 5000 analyzer Haemonetics America . In addition, blood lipids parameters were detected using an OLYMPUS AU5800 biochemical analyser. The instruction manual was referenced for specific protocols.

Statistical analysis
Normally distributed data were expressed as median plus or minus standard deviation and one-way anova was performed between groups. Percentage data were expressed as percentage (%) and χ2 test was performed between groups. The pvalues were corrected for the number of comparisons using the Bonferroni method, and all tests were two-tailed. Sperson correlation and multivariate logistic regression model were selected to analyze the correlation between each index and CIMT. Receiver operating characteristics (ROCs) were analyzed to determine the optimal cut-off values, and the area under the curve (AUC) values were compared to select the variables that predict the differentiation. P<0.05 was considered statistical significant. All statistical analysis was performed using the Statistical   Table 1 and Figure 1).

Detection of carotid AS in the 3 groups
In the hypertension combined with cerebral infarction group, 80 patients had carotid intima thickening, and 51 patients had plaque formation; therefore, there were 131 patients with carotid AS, accounting for 68.23% of the group. In the hypertension group, 80 patients had carotid intima thickening, and 23 patients had plaque formation; therefore, there were 103 patients with carotid AS, accounting for 53.37% of the group. In the control group, 23 patients had carotid intima thickening, and 13 patients had plaque formation; therefore, there were 36 patients with carotid AS, accounting for 23.84% of the group. Analysis using the χ2 test showed that among the 3 groups, those with normal carotid intima, intima thickening, and plaque formation differed significantly (χ2=16.162, p<0.001). The percentage of AS increased significantly in the hypertension combined with cerebral infarction group (χ2=67.702, p<0.001) ( Table 2). .003). After adjusting for age, sex, smoking and blood lipid, the correlation was still found (Table 3 and Figure 2). The results showed that Lp-PLA2, Hcy, R were linearly correlated with CIMT after gradual correction of age, sex, smoking and blood lipid by stepwise linear regression analysis of multiple factors. In addition, the VIF (Variance Inflation Factor) values of each variable in the model are less than 2, indicating that there is no collinearity among the variables ( Table 4).

Correlation analysis between plasma
Analysis of the correlation among plasma Lp-PLA2, Hcy, CRP, Lp(a), AT-III, and TEG parameters in all enrolled subjects showed that Lp-PLA2 positively correlated with Hcy, α-angle, MA, A, CI, and G and negatively correlated with AT-III and K. Hcy positively correlated with CRP, Lp(a), α-angle, MA, A, CI, and G and negatively correlated with AT-III, R, K, and EPL (Table 5).
2.5 Diagnostic value of Lp-PLA2, Hcy, CRP, Lp(a), AT-III, or TEG alone and in combination for the determination carotid AS risk.
The ROC curve showed that in the evaluation of carotid AS risk using Lp-PLA2, the AUC was 0.677, the 95% CI was 0.632 to 0.722, and the threshold value was 140.01 ng/ml; in the evaluation of carotid AS risk using Hcy, the AUC was 0.707, the 95% CI was 0.663 to 0.750, and the threshold value was 12.79 μmol/L; in the evaluation of carotid AS risk using Lp(a), the AUC was 0.557, the 95% CI was 0.509 to 0.606, and the threshold value was 12.50 mg/L; and in the evaluation of carotid AS risk using AT-III, the AUC was 0.555, the 95% CI was 0.506 to 0.604, and the threshold value was 94.5%. The TEG-related parameters that were significant in the evaluation of carotid AS risk were R, K, α-angle, MA, CI, and G; their AUCs were 0.600, 0.621, 0.624, 0.562, 0.555, and 0.562, respectively, and the threshold values were 6.95 min, 1.65 min, 65.95°, 56.75 mm, 1.75, and 6551 d/sc, respectively. For the determination of carotid AS risk using the combination of all TEG parameters, the AUC was 0.663, and the 95% CI was 0.617 to 0.708. For the determination of carotid AS risk using the combination of Lp-PLA2, Hcy, CRP, Lp(a), and AT-III, the AUC was 0.747, and the 95% CI was 0.706 to 0.787. For the determination of carotid AS risk using the combination of all items, the AUC was 0.759, and the 95% CI was 0.719 to 0.798; the AUC was the largest (Table 7 and Figure 3).

Conclusions
Hcy and Lp-PLA2 were better diagnostic indicators in the prediction of carotid AS risk The pathology of AS involves chronic inflammation with slow progression. Various inflammatory mediators and chemokines participate in its development and progression (20,21). The stability of AS plaques is key to determining disease prognosis (22). The rupture of AS plaques can cause a series of serious lifethreatening consequences such as cerebral infarction or myocardial infarction. The development and progression of AS is closely associated with oxidative stress, inflammation, and dyslipidaemia (23). The formation of foam cells from mononuclear macrophages through a series of pathological processes involving the phagocytosis of lipids under the arterial intima has been recognized as a marker of the development and progression of AS plaques (24). Abnormal autophagy in macrophages and macrophages themselves play a pivotal role in the progression of AS plaques (25). After autophagy, macrophages can release Lp-PLA2 to promote foam cell formation, which is closely associated with plaque instability (26).
Abnormal autophagy in macrophages is also closely associated with the process of inflammation (27). Autophagy can directly regulate the production and secretion of inflammatory factors such as IL-1β. In addition, inflammatory factors released by mononuclear macrophages also play an autophagy regulatory role (28, 29).
In mediators associated with carotid AS, Lp-PLA2 can hydrolyse oxidized LDL in the arterial wall to produce lysophosphatidylcholine and oxidized free fatty acids. These R value was also a better diagnostic indicator for the prediction of carotid AS In the development of AS, deregulation the coagulation and anti-coagulation system in the body also plays a very important role. The human body as a whole relies on the collaboration among multiple systems and multiple tissues. Mediators and systems never work in isolation. The complement system, inflammatory system, and coagulation system influence and regulate one another during the development of AS. AT-III is closely associated with the hypercoagulable state, and a reduction in its activity suggests thrombosis. It has been shown that a reduction in AT-III activity might predict the progression of acute coronary events in patients with coronary artery disease; in addition, the level of reduction in AT-III activity positively correlated with coronary artery stenosis (37, 38). This study showed that the AT-III levels in the hypertension combined with cerebral infarction group and the simple hypertension group were not statistically different and that the AT-III levels in the two groups were much lower than corresponding indicators in the control group.
These results indicated that patients with hypertension were already in a thrombophilic state whether or not they had combined cerebral infarction. The correlation analysis results showed that AT-III did not correlate with AS. The ROC curve analysis showed that AT-III was not suitable for the diagnosis of carotid AS.

Ethics approval and consent to participate
The study was approved by the Ethical Committee of Affiliated Hospital of Nanjing University of Traditional Chinese Medicine. All subjects agreed the study and signed informed consent letters.

Consent to publish
The manuscript is approved by all authors for publication.

Availability of data and materials
The data and materials are available.

Competing interests
The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

Funding
The manuscript is funded by National Natural Science Foundation of China(NO: 81603358).  Tables   Tables   Tab.1    Combined-a combined the parameters of R K α-Angel MA EPL LY30 A CI G Combined-b combined the paremeters of Lp-PLA2 Hcy CRP Lp(a) AT-Ⅲ Combinedc combined the paremeters of Lp-PLA2 Hcy CRP Lp(a) AT-Ⅲ R K α-Angel MA EPL LY30 A CI G. Figure 1 Comparison of the levels of Lp-PLA2 Hcy CRP Lp(a) AT-Ⅲ TEG and CIMT in three groups Gro Figure 2 Analysis of the correlation of Lp-PLA2 Hcy CRP Lp(a) AT-Ⅲ TEG and CIMT Figure 3 3 The ROC analysis of combined biomarkers and individual biomarker in the evaluation of car