Increased plasma lipoprotein-associated phospholipase A2 levels are associated with coronary slow flow

Objective Coronary slow flow (CSF) is characterized by delayed opacification of distal epicardial coronary arteries without significant coronary stenosis. In addition, The changes of lipoprotein-associated phospholipase A2 (Lp-PLA2) as a significant predictive factor for CSF remain controversial. The study aims to investigate the association between plasma Lp-PLA2 and CSF. Methods In this retrospective study, 170 consecutive patients who underwent coronary angiography were enrolled in Beijing Anzhen Hospital from January 2017 to September 2019, and were divided into CSF group and normal control groups. According to coronary blood flow rate measured by the thrombolysis in myocardial infarction frame count (TFC) method, CSF was defined as TFC > 27. Serum Lp-PLA2 levels were measured in an enzyme-linked immunosorbent assay. Results Lp-PLA2 levels were higher in the CSF group than in the control group (288.6 ± 50.3 versus 141.9 ± 49.7, P < 0.001) and were significantly correlated with the mean coronary artery thrombolysis in myocardial infarction (TIMI) frame count (r = 0.790, P<0.001). Logistic regression analysis showed that high Lp-PLA2 was independently associated with CSF after adjustment for conventional risk factors (OR = 1.040, CI = 1.022–1.059, P<0.001). Male sex (OR = 2.192, CI = 1.161–4.140, P = 0.016) and hypertension (OR = 1.965, CI = 1.034–3.736, P = 0.039) were also CSF risk factors. Receiver-operating characteristic curve (ROC) analysis showed that Lp-PLA2 levels can predict CSF severity; the predictive power was higher than the other risk factors. Conclusion Our study demonstrated that patients with CSF had higher circulating levels of Lp-PLA2 than normal controls. After adjustment for potential confounders, increased Lp-PLA2 was independently associated with presence of CSF.

The coronary slow flow (CSF) phenomenon is a special phenomenon involving coronary microcirculation dysfunction, which is characterized by delayed distal vascular opacification without significant epicardial coronary stenosis. Studies have shown that slow blood flow is often associated with adverse cardiovascular events, including angina, myocardial infarction, malignant arrhythmias and even sudden cardiac death, suggesting a poor prognosis [1]. Although it has been 40 years since Tambe et al. first discovered slow blood flow [2], the exact mechanism of its pathogenesis remains unclear. A large number of studies have shown that the pathogenesis of CSF may involve vascular endothelial dysfunction, vascular inflammation, obesity, atherosclerosis and other factors [3][4][5]. However, there is a lack of effective biomarkers that specifically predict CSF. Recently, a vast majority of evidence has indicated that many inflammatory mediators such as interleukin-1, interleukin-10, and the lymphocyte to monocyte ratio, are associated with the pathogenesis of CSF, indicating the presence of a proinflammatory process occurring as a result of the phenomenon [6][7][8]. Thus, it is of value to identify the risk factors for CSF in order to detect and prevent CSF as early as possible.
Lipoprotein-associated phospholipase A2 (Lp-PLA 2 ), a leukocyte-derived enzyme, is involved in the metabolism of low-density lipoprotein (LDL), propagating atherogenesis, and mediating the inflammatory process of the vascular wall [9]. Lp-PLA 2 circulates in plasma in its active form with complexes of LDL and high density lipoprotein (HDL) [10]. Recent evidence suggests that Lp-PLA 2 plays an important role in the pathophysiology of atherosclerosis and as a predictive biomarker for predicting future cardiovascular events [11]. According to its properties of proinflammation and atherogenesis, and the fact that increased Lp-PLA 2 has been found to be closely associated with inflammation and atherosclerosis, we speculate that Lp-PLA 2 may be associated with CSF [12]. Therefore, in this study, our aim was to evaluate the relationship between coronary blood flow and the Lp-PLA2 level.

Study patients
The retrospective study was carried out from January 2017 to September 2019 at Anzhen Hospital. A total of 170 patients who underwent coronary angiography with clinical chest pain and without significant coronary stenosis were consecutively enrolled in the observational study; 78 of the patients had slow coronary flow without any stenosis evidenced by coronary angiography, and 92 patients had normal coronary arteries and normal flow. Patients with prior evidence of coronary artery disease (acute coronary syndrome, coronary interventions history, coronary plaque and significant atherosclerotic lesions (stenosis above 40%), coronary ectasia, coronary calcification), congenital heart disease, valvular heart disease, cardiomyopathy, ischemic electrocardiogram, hematological system disease, tumors, heart failure, liver dysfunction, or kidney dysfunction or who had recently undergone surgery were excluded from the study. The study was approved by the Beijing An zhen Hospital Ethics Committee of Capital Medical University, and all patients provided informed consent.

Anthropometric and laboratory measurements
The study measured demographic characteristics, including age, sex, height, weight, body mass index (BMI) and waist circumference. Smoking, hypertension, diabetes mellitus and medication use were collected from electronic medical records. After fasting for at least 8 h, peripheral blood was collected on the morning of the first day of admission and stored at − 70°C. The plasma Lp-PLA 2 activity was measured using an enzyme-linked immunoassay (PLAC™ test, DIADEXUS, USA). Glucose, urea, creatinine, total cholesterol (TC), triglyceride (TG), HDL, and LDL levels were measured using a chemiluminescence method with a Roche Diagnostics Cobas analyzer Cobas 8000, c702 module. High sensitivity Creactive protein (hs-CRP) levels were also measured by the nephelometric method. All samples were tested in triplicate according to the manufacturer's protocols.

Diagnostic criteria
ACS was diagnosed according to European Society of Cardiology guidelines in 2015 [13]. Cardiomyopathy was diagnosed by established histological, immunological, and immunohistochemical criteria according to the WHO classification [14]. The diagnostic criteria of diabetes mellitus was according to WHO guidelines or by indication for insulin or anti-diabetic medications [15]. Hypertension was defined as repeated systolic pressure ≥ 140 mmHg and/or diastolic pressure ≥ 90 mmHg at least twice, or previously diagnosed hypertension [16].

Coronary angiography protocols
CSF was defined according to the thrombolysis in myocardial infarction (TIMI) frame count (TFC) method. Coronary angiogram was performed using a digital subtraction angiography system (Allura Xper FD20; Philips Medical Systems, Best, the Netherlands), which used the standard Judkins technique to obtain images at the rate of 30 frames/s. The injection rate was 4-5 ml/s in the left coronary artery and 3-4 ml/s in the right coronary artery (RCA). The left coronary artery was injected with 8-10 ml contrast agent and RCA was injected with 6-8 ml contrast agent. The left anterior descending coronary artery (LAD) was imaged as the right anterior oblique projection with an angle of 20-25°, the left circumflex coronary artery (LCX) was imaged as the right anterior oblique projection with an angle of 20-25°, and the RCA was imaged as the positive projection with an angle of 30°. The first frame was defined when the contrast agent touched two medial walls of the coronary artery and advanced steadily with a diameter of more than 70%, and the last frame was defined when the leading edge of the contrast agent reached the end of the branches of the coronary artery. The frame counts in the LAD were divided by a factor of 1.7 to correct for its longer length. According to Gibson et al., any frame count over 27 is considered abnormal and indicates significant CSF [17]. All patients were carefully monitored for pulse and blood pressure during coronary angiogram.

Statistical analysis
Data were analyzed with IBM SPSS software version 23.0. Categorical variables were presented as frequencies

Patient characteristics
The baseline clinical characteristics of the study participants are shown in Table 1. We enrolled 170 patients (mean age 61.6 ± 9.7, 60.6% male) who underwent coronary angiography; the sample was composed of 78 patients (mean age 60.2 ± 9.7, 70.5% male) with CSF and 92 patients (mean age 62.7 ± 9.5, 52.1% male) with normal coronary flow (NCF). As shown in Table 1 Fig. 1). The TFC values of the two groups of coronary arteries were calculated respectively. The mean TFCs of LAD, LCX and RCA were significantly higher in CSF patients than in NCF patients (p < 0.001 for each coronary artery, Table 2, Fig. 2).

Discussion
As far as we know, the present study demonstrated for the first time that there was an independent relationship between Lp-PLA 2 and CSF phenomenon. The major    finding was that Lp-PLA 2 levels were positively correlated with CSF and significantly elevated in subjects with CSF. In addition, multivariate logistic regression analysis and ROC curves showed that Lp-PLA 2 was still independently associated with the presence and severity of CSF. Male sex and hypertension also independently predicted the presence of the CSF phenomenon. Although CSF has been found to be related to microvascular diseases, vascular endothelial dysfunction, coronary atherosclerosis, oxidative stress, insulin resistance, adipocytokines, and abnormal blood composition, the exact mechanisms are still unknown [18][19][20]. Tambe proposed that the CSF phenomenon might be related to abnormal microcirculation in 1972 [2]. Oğuzhan Çelik et al. identified a correlation between the extent of disruption in endothelial function and the CSF level using the flowmediated dilation (FMD) method [21]. Several studies have found that the imbalance between endothelin-1 and nitric oxide in patients with CSF supports the involvement of endothelial dysfunction in CSF etiopathogenesis [22]. An intravascular ultrasound study demonstrated that diffuse coronary calcification was present in 88% of CSF patients, and statin therapy has been shown to improve coronary flow in CSF subjects [23]. Decreased fractional flow reserve in CSF patients has been demonstrated by intravascular ultrasound (IVUS) to result in increased resistance in the epicardial coronary artery due to diffuse atherosclerotic disease [24].
The role of chronic inflammation-mediated endothelial injury in CSF has been widely recognized. Muhammed Oylumlu et al. revealed that the platelet-to-lymphocyte ratio could be an important risk factor in patients with CSF [25]. Li et al. showed that plasma CRP and IL-6 as inflammatory markers were positively correlated with TIMI frame count in patients with CSF [26]. In our study, Lp-PLA 2 was shown to be an independent predictor of CSF. Lp-PLA 2 , known as platelet-activating factor acetylhydrolase, is a calcium-independent serine lipase [10]. Lp-PLA 2 is synthesized by macrophages and other inflammatory cells and circulates in the human blood mainly with LDL granules (80-85%) and less often with HDL [9]. Lp-PLA 2 is involved in the oxidative modification of vascular wall LDL to generate oxidized phospholipids and oxidized non-esterified fatty acids, which can promote the development of vascular inflammation and atherosclerotic plaques [27]. Lp-PLA 2 has been identified as an independent predictor of cardiovascular disease (CVD),and has been recommended as an adjunct to traditional risk assessment in patients with moderate and high 10-year risk of CVD as defined by Framingham risk scores [28]. In the REGARDS study, Lp-PLA 2 activity was associated with coronary heart disease (CHD) risk over 5.3 years based on Cox proportional hazards regression [29]. Our study found that in the CSF group, with the increase in TFC, Lp-PLA2 levels exhibited an increasing trend, and there was a significant positive correlation between CSF and Lp-PLA 2 . Additionally multivariate logistic regression analysis showed that Lp-PLA 2 could independently predict CSF. However, the mechanisms underlying the relationship between Lp-PLA 2   Fig. 1; model 3: model 2 + medicine and CSF are not entirely understood. The most reliable hypothesis seems to be that CSF is caused by increased microvascular resistance induced by endothelial dysfunction due to Lp-PLA 2 mediated chronic inflammation. Numerous studies have attempted to define the demographics, characteristics and independent predictors of patients with CSF. Arbey Y found that current smoking was associated with CSF [30]. Other studies have shown that BMI and hypertension were predictors of CSF phenomena [31]. Compared to other studies, our study did not find an association between BMI and CSF. Our study also shows that Lp-PLA 2 , male gender,c and hypertension are independent risk factor of the presence of CSF phenomena. Tsimikas found in his study that there was a significant correlation between Lp-PLA 2 and LDL, non-HDL, and HDL [11]. However, we did not find any difference in LDL between the two groups, which may be related to the use of lipid-lowering drugs.

Limitations
Our study has some limitations: (1)This study was limited by a small and nonrandomized sample, which may restrict the generalizability of our results. (2)The study did not examine the ethnic background or socioeconomic status of the study population.(3)This study was also a single center, retrospective study. Multicenter randomized controlled trials must be conducted to explore the relationship between Lp-PLA 2 and CSF. (4)The use of drugs, such as lipid-lowering drugs, may affect the results of experiments. (5)Finally, we determined only the mass of Lp-PLA 2 instead of its activity due to our laboratory limitations.

Conclusion
In summary, the main finding was that Lp-PLA 2 levels were positively correlated with CSF. The multivariate regression results showed that Lp-PLA 2 was independently associated with the presence and severity of CSF. Currently, there is still no effective method to treat CSF. This study indicated the association between Lp-PLA 2 and CSF, which may provide a new therapeutic target for the treatment of CSF. However, the exact pathophysiological mechanisms of Lp-PLA 2 in CSF require further study to elucidate.