Plasma Levels of Receptor Interacting Protein Kinase-3 Correlated with Coronary Artery Disease
Introduction
Coronary artery disease (CAD) remains a leading cause of mortality worldwide, particularly in developed countries. Despite significant advancements in understanding the pathogenesis of atherosclerosis and CAD development, critical questions persist. Firstly, there is a pressing need for novel biomarkers to assist in screening CAD subtypes based on current definitions. Secondly, more convenient and non-invasive methods are required to evaluate the severity of coronary artery atherosclerosis, as alternatives to invasive procedures like coronary computed tomography angiography and coronary arteriography. Additionally, identifying patients at risk of acute coronary syndrome (ACS) from others is crucial for timely prevention and treatment.
Emerging evidence suggests that necroptosis, a form of programmed cell death, plays a significant role in various inflammatory conditions and diseases, including human atherosclerosis and atheroma development. Receptor interacting protein kinase-3 (RIP3) is a critical regulator of necroptosis. Circulating RIP3 levels have been considered important markers in diagnosing and predicting diseases such as sepsis, acute kidney injury, and ST-segment elevation myocardial infarction (MI). However, the association between RIP3 levels and CAD requires further confirmation in large-scale studies. Moreover, the differences between plasma RIP3 levels and serum RIP3 levels have not been thoroughly discussed.
This study aimed to confirm the relationship between plasma RIP3 levels and CAD. Furthermore, it sought to explore the relationship between plasma RIP3 levels and various CAD subtypes and severity.
Methods
Ethical Approval
The study was approved by the Ethics Committee of Peking Union Medical College Hospital and conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.
Study Population
From September 2017 to January 2018, Chinese patients with CAD aged between 30 and 100 years who were hospitalized for coronary angiography at Peking Union Medical College Hospital were consecutively recruited. Patients with ≥50% stenosis in ≥1 main coronary artery were diagnosed with CAD. Patients with CAD were further divided into three subgroups: stable coronary artery disease (SCAD), unstable angina (UA), and MI. They were also categorized according to the number of pathological branches involved. Chinese volunteers free from a known history of CAD were recruited as controls. Subjects with acute renal injury, sepsis, or shock were excluded from the study.
UA was defined as a normal measurement of cardiac troponin with at least one of the following criteria: prolonged (>20 minutes) angina pain at rest, new onset angina (Class II or III according to the Classification of the Canadian Cardiovascular Society), recent destabilization of previously stable angina with at least Canadian Cardiovascular Society Class III angina characteristics (crescendo angina), or post-MI angina. SCAD was defined as angina that did not fulfill the above UA criteria. MI was defined as a rise and/or fall of cardiac troponin with at least one value above the 99th percentile upper reference limit and with at least one of the following: ischemia symptoms; new or presumed new significant ST-segment-T wave changes or new left bundle branch block; development of pathological Q waves in the electrocardiogram; imaging evidence of new viable myocardium loss or new regional wall motion abnormality; and identification of an intra-coronary thrombus by angiography or autopsy.
Coronary Angiography and Image Interpretation
Coronary angiography was performed with a conventional angiography unit (Integris H; Philips Medical Systems, Amsterdam, the Netherlands). Coronary artery stenosis was imaged in the center of the field from multiple projections. Using the quantitative coronary analysis system, the coronary atherosclerotic lesion severity was evaluated from at least two projections by two interventional cardiologists with at least 5 years of experience who were blinded to the patients’ clinical information. Coronary angiographies were performed to determine the severity of atherosclerosis by the Gensini score (GSS).
Plasma/Serum Collection and Plasma/Serum RIP3 Level Quantification
For patients with MI, peripheral blood was drawn from the radial or femoral artery before coronary angiography or from the vein in the morning on the day after admission according to their disease subtypes and treatment guidelines. For patients with SCAD and UA, peripheral fasting blood was drawn from the vein in the morning the day after admission. For controls, peripheral fasting blood was drawn from the vein in the morning. Plasma blood was centrifuged at 1500 r/min for 10 minutes at 20°C, and the supernatant was collected. Serum blood was centrifuged at 1000 r/min for 10 minutes at 20°C, and the supernatant was collected. They were collected into sodium heparin tubes (BD) and stored at -80°C within 4 hours until RIP3 levels were tested. Plasma/serum RIP3 levels were detected with ELISA kits (Cusabio, Wuhan, China) according to the manufacturer’s instructions.
Other Parameters
Subjects’ body mass index was calculated as weight (kg) divided by height squared (m2). Blood pressure was measured with a standard mercury sphygmomanometer by specially trained nurses with the patient in a seated position after ≥5 minutes of rest. Hypertension and diabetes were diagnosed using current guidelines. Peripheral venous blood was taken from the antecubital vein early in the morning while the patient was in a fasting state and before any medications were administered. The levels of each lipid profile component (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides), creatinine, and high-sensitivity C-reactive protein were measured.
Statistical Analysis
The Shapiro-Wilk test was used to determine normality of the data distribution for sample n < 50. The Kolmogorov-Smirnov test was used to determine normality of the data distribution for sample n ≥ 50. Normally distributed data were expressed as the mean ± standard deviation, and data with non-normal distributions were expressed as the median (Q1, Q3). Continuous, normally distributed variables between two groups were analyzed by Student t-test. The Mann-Whitney U test was applied for data of this type that were not normally distributed. Continuous, normally distributed variables among four groups were analyzed by a one-way analysis of variance. The Kruskal-Wallis H test was applied for data of this type that were not normally distributed. Categorical variables were summarized as count and percentage and were compared by the Chi-square test. Logistic regression was used to determine the relationship between plasma RIP3 levels and CAD after adjusting for confounding factors. The Youden’s index in the receiver operating characteristic (ROC) curves was used to determine the optimal cut-off of plasma RIP3 levels for detecting CAD. Plasma RIP3 levels were compared among controls and patients in different subtypes of CAD using the analysis of variance type of trend analysis. To explore the relationship between plasma RIP3 levels and proportion of cases of ACS in subjects, all subjects were divided into four categories according to the quartiles of plasma RIP3 levels. The correlation between plasma RIP3 and GSS was calculated using multiple linear regression models and adjusted for confounding factors. A P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS Statistics software, version 22.0 (SPSS Inc., Chicago, IL, USA).
Results
Participant Characteristics
A total of 484 subjects were enrolled in this study, including 166 controls and 318 patients with CAD (patients with SCAD, n = 93; patients with UA, n = 153; and patients with MI, n = 72). The mean ages were 60.1 ± 9.4 years for controls and 64.4 ± 10.8 years for patients with CAD. Traditional cardiovascular risk factors and New York Heart Association classes in the study population are summarized in Table 1. Most patients with CAD were male and more likely to have a history of hypertension and diabetes. CAD groups exhibited relatively higher high-sensitivity C-reactive protein levels compared with the controls.
Differences Between Plasma and Serum RIP3 Levels
Both plasma and serum levels of RIP3 were tested in 23 controls, 20 patients with SCAD, 20 patients with UA, and 20 patients with MI. The median levels of plasma RIP3 were significantly higher than corresponding serum levels in total (433.75 [296.28, 516.11] pg/mL vs. 39.20 [20.43, 65.33] pg/mL, Z = 257.000, P < 0.001) and in each group (controls: 244.28 [171.34, 287.03] pg/mL vs. 24.37 [16.26, 39.44] pg/mL, Z = 0.000, P < 0.001; patients with SCAD: 426.98 [395.70, 493.35] pg/mL vs. 34.67 [22.00, 51.31] pg/mL, Z = 0.000, P < 0.001; patients with UA: 464.98 [455.20, 512.51] pg/mL vs. 30.21 [16.08, 61.10] pg/mL, Z = 0.000, P < 0.001; patients with MI: 551.34 [473.64, 656.40] pg/mL vs. 105.23 [59.68, 178.03] pg/mL, Z = 0.000, P < 0.001) [Figure 1].
Biomarker for CAD and Plasma RIP3 Cut-off Level
We tried to determine if plasma levels of RIP3 could potentially serve as a marker for CAD. We expanded the sample size of RIP3 plasma level testing to include all 318 patients with CAD and 166 controls. The median levels of plasma RIP3 in patients with CAD were significantly higher than that of controls (406.87 [311.51, 516.59] pg/mL vs. 241.61 [175.83, 318.13] pg/mL, Z = 9565.000, P < 0.001). To diminish the confounding effects of traditional CAD risk factors, we conducted a logistic regression analysis. The Youden’s index in the ROC curve was used to determine the optimal cut-off of plasma RIP3 levels for detecting CAD. According to the ROC curve analysis, the optimal cut-off value for plasma RIP3 levels in predicting CAD was found to be 324.51 pg/mL, with a specificity of 80.0% and sensitivity of 73.0% (area under curve = 0.819, 95% confidence interval = 0.779–0.859) [Figure 2]. After adjusting for confounding factors, plasma RIP3 levels still remained strongly associated with CAD (P < 0.001) [Table 2].
Association Between Plasma RIP3 Levels and CAD Severity
Plasma RIP3 levels increased linearly from controls (241.61 [175.83, 318.13] pg/mL), to patients with SCAD (388.39 [328.8, 501.66] pg/mL), then patients with UA (386.91 [303.14, 478.67] pg/mL), and finally to patients with MI (455.04 [343.36, 607.44] pg/mL; P for linear trend < 0.001) [Figure 3]. We did not find a similar linear relationship in plasma RIP3 levels when comparing patients with normal coronary arteries (245.24 [177.47, 319.58] pg/mL), single vessel disease (403.53 [311.99, 505.46] pg/mL), double vessel disease (416.56 [297.74, 500.04] pg/mL), and triple vessel disease (401.26 [311.09, 521.91] pg/mL) [Supplemental Figure 1]. However, there were significant differences in plasma RIP3 levels between the subjects with normal coronary arteries and patients with different pathological branches involved.
To explore the relationship between plasma RIP3 levels and proportion of cases of ACS in subjects, all subjects were divided into four categories according to which quartile their plasma RIP3 level fell into. Clinical characteristics are listed in Supplementary Table 1. Subjects with higher plasma RIP3 levels were older and were more likely to suffer from comorbidities. Moreover, they had higher levels of high-sensitivity C-reactive protein. Notably, we found a significant positive correlation between proportion of cases of ACS in subjects and the plasma RIP3 level quartile [Supplementary Figure 2].
We adopted multiple linear regression models to determine the correlation between plasma RIP3 levels and GSS. Plasma RIP3 levels were associated with GSS after adjustments for traditional cardiovascular risk factors (P < 0.05) [Table 3].
Discussion
This study confirmed that in patients with CAD, plasma RIP3 levels were significantly higher than controls and they were strongly associated with CAD, even after adjusting for confounding factors. We believed that plasma RIP3 is an independent CAD risk factor and might be regarded as a surrogate marker for CAD screening. Plasma RIP3 levels were also found to be positively correlated with coronary arterial atherosclerosis severity and its clinical manifestations. This indicated that plasma RIP3 could be used to predict disease severity.
The human RIP3 gene is located on chromosome 14, and its messenger RNA encodes a polypeptide of 518 amino acids. Several studies have pointed out that RIP3 is essential for the execution of tumor necrosis factor-induced necroptosis downstream of RIP1. In addition, through the analysis of RIP3 deficient mice, researchers have implicated the role of necroptosis in atherosclerosis, alcoholic liver disease, and retinal degeneration. The critical pro-necroptotic factor RIP3 is found to be present and activated in human atherosclerotic plaque. RIP3 deficient, atherosclerosis-prone low-density lipoprotein receptor-deficient, or apolipoprotein E-deficient mice had a significant reduction in advanced atherosclerotic lesions. In addition, in vitro cellular studies showed that RIP3 deletion prevented primary necrosis of macrophages. These findings indicated a driver function of RIP3 in atherosclerotic necrosis. In the present study, plasma RIP3 levels were associated with GSS. However, the extent to which necroptosis and its mediator RIP3 play a role in atherosclerosis is unknown.
It is notable that we demonstrated a significant association between plasma RIP3 level quartiles and prevalence of ACS cases, which might shed light in predicting adverse events in patients with CAD. In 1989, Muller et al proposed the concept of “vulnerable plaque.” Predominantly derived from pathological observations at autopsies, the “plaque-rupture” hypothesis and new views of vulnerable atherosclerotic coronary plaque features were established. Acute coronary thrombosis and subsequent acute MI are now regarded as the results of sudden plaque rupture or erosion. And efforts have been made to identify high-risk patients using advanced imaging methods to find “vulnerable” coronary sites. However, these methods have many disadvantages, such as price, inconvenience, and poor efficacy. Karunakaran et al found that necroptotic cell death in humans was activated in advanced atherosclerotic plaques and expression of RIP3 and mixed lineage kinase domain-like protein was increased with unstable carotid atherosclerosis. This indicated that RIP3 might correlate with lesion vulnerability. The elevated plasma RIP3 levels we found in patients with MI might be due to the rupture of atherosclerotic plaques with their subsequent release into circulating plasma. This finding might also be related to thrombus formations in the coronary artery. Zhang et al demonstrated a role for RIP3 in promoting in vivo thrombosis and hemostasis. RIP3 inhibitors dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo.
In this study, we found that plasma levels of RIP3 were significantly higher than serum levels of RIP3 in human patients. Circulating RIP3 levels have been considered important markers in diagnosing and predicting diseases. Kashlov et al figured out that levels of serum RIP3 were not altered significantly in patients with ST-segment elevation MI, compared to controls. However, in our study, we pointed out that plasma levels of RIP3 were significantly higher than the corresponding serum levels. Zhang et al confirmed that RIP3 was expressed in platelets, and the differences between serum and plasma values might be influenced by the platelets at the time of blood coagulation.
Some limitations of our study should also be considered. This study was a cross-sectional study and it was impossible to assess variables’ contributions over time. Therefore, further studies, utilizing a prospective cohort, are needed to confirm the association between plasma RIP3 levels and CAD. The underlying mechanism behind our findings remains uncertain, since the source of RIP3 detected in plasma is still unclear. Additionally, participants in our study came from China; thus, generalizability to other ethnic or racial populations may not be valid. Similar research is necessary in other regions to assess the general application of our findings.
In conclusion, the results of this study confirmed that compared to serum, plasma RIP3 levels were detected in significantly higher concentrations. Plasma RIP3 could be used as a surrogate marker for CAD screening or a predictor for the severity of CAD progression. Furthermore, plasma RIP3 levels were positively associated with CAD subtypes and proportion of cases of ACS in subjects. Accordingly, elevated plasma RIP3 levels may indicate the need for a careful evaluation for the presence and progression of CAD.
doi.org/10.1097/CM9.0000000000000225
Was this helpful?
0 / 0