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Plasma levels of receptor interacting protein kinase-3 correlated with coronary artery disease

Plasma levels of receptor interacting protein kinase-3 correlated with coronary artery disease Background: Necroptosis plays an important role in human atherosclerosis and atheroma development. Since receptor interacting protein kinase-3 (RIP3) acts as a key mediator of necroptosis, this study aimed to explore its relationship between plasma RIP3 levels and coronary artery disease (CAD) and discover a potential new biomarker for screening CAD subtypes and severity. Methods: A total of 318 patients with CAD who had coronary angiography and 166 controls in Peking Union Medical College Hospital from September 2017 to January 2018 were enrolled in this study. Patients with CAD were divided into three subgroups: patients with stable coronary artery disease (SCAD), patients with unstable angina (UA), and patients with myocardial infarction (MI). The severity of atherosclerosis was determined by Gensini score (GSS). Logistic regression was used to determine the relationship between plasma RIP3 levels and CAD. The correlation between plasma RIP3 and GSS was calculated using multiple linear regression models. Results: Overall, plasma RIP3 levels were significantly higher than serum RIP3 levels. Plasma RIP3 levels in patients with CAD were significantly higher than those in controls. Plasma RIP3 levels were strongly associated with CAD (odds ratio: 6.00, 95% confidence interval 3.04–11.81; P < 0.001). Plasma RIP3 levels increased linearly from controls to patients with SCAD, then patients with UA, and finally to patients with MI. We found a significantly positive correlation between proportion of cases of acute coronary syndrome in subjects and their plasma RIP3 level quartile. Plasma RIP3 levels were also associated with GSS (B 0.027; standard error 0.012; P < 0.05). Conclusions: Plasma RIP3 levels were independently associated with CAD. Plasma RIP3 levels could potentially supplement clinical assessment to screen CAD and determine CAD severity. Keywords: Receptor interacting protein kinase-3; Coronary artery disease; Necroptosis; Atherosclerosis; Acute coronary syndrome; Biomarker Introduction Emerging pieces of evidence have suggested that necrop- tosis might play an important role in several inflammatory Coronary artery disease (CAD) is common in developed conditions and diseases, including human atherosclerosis [1,2] countries and remains the leading cause of death. A [4] and atheroma development. Receptor interacting pro- study has focused on the pathogenesis of atherosclerosis tein kinase-3 (RIP3) is currently regarded as a critical [3] and the development of CAD. However, several [5] regulator of programmed necrosis/necroptosis. As far as questions remain unanswered. First, it would be better if we know, circulating RIP3 levels have been considered new biomarkers could be found to assist in screening CAD important markers in diagnosing and predicting some subtypes based on current definitions. Second, we need [6] [7] diseases, including sepsis, acute kidney injury, and ST- more convenient and non-invasive methods to evaluate the [8] segment elevation myocardial infarction (MI). It was coronary artery atheromatous severity instead of coronary reported that the levels of serum RIP3 were not altered computed tomography angiography and coronary arteri- significantly in patients with ST-segment elevation MI ography. Moreover, it is hard but important to select [8] compared to controls. However, the association between patients who might be suffering with acute coronary RIP3 levels and CAD required further confirmation in a syndrome (ACS) from others so that appropriate and timely prevention and treatment can be given. Xiao-Min Hu and Xi Chen contributed equally to this study. Access this article online Correspondence to: Prof. Shu-Yang Zhang, Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Quick Response Code: Medical College, No.1 Shuaifuyuan, Dongcheng District, Beijing 100730, China Website: E-Mail: shuyangzhang103@163.com www.cmj.org Copyright © 2019 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license. This is an open access article distributed under the terms of the Creative DOI: Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is 10.1097/CM9.0000000000000225 permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Chinese Medical Journal 2019;132(12) Received: 07-01-2019 Edited by: Xin Chen Downloaded from http://journals.lww.com/cmj by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/01/2020 Chinese Medical Journal 2019;132(12) www.cmj.org large-scale study. And the differences between plasma projections. Using the quantitative coronary analysis RIP3 levels and serum RIP3 levels have not been discussed. system, the coronary atherosclerotic lesion severity was evaluated from at least two projections by two interven- The aim of this study was to confirm the relationship tional cardiologists with at least 5 years of experience who between plasma RIP3 levels and CAD. Furthermore, we were blinded to the patients’ clinical information. Coro- tried to explore the relationship between plasma RIP3 nary angiographies were performed to determine the [12] levels and various CAD subtypes and severity. severity of atherosclerosis by the Gensini score (GSS). Methods Plasma/serum collection and plasma/serum RIP3 level quantification Ethical approval For the patients with MI, peripheral blood was whether This study was approved by the Ethics Committee of drawn from the radial or femoral artery before coronary Peking Union Medical College Hospital and conducted angiography or from the vein in the morning on the day according to the principles of the Declaration of Helsinki. after admission according to their disease subtypes and Written informed consent was obtained from all treatment guidelines. For the patients with SCAD and UA, participants. peripheral fasting blood was drawn from the vein in the morning the day after admission. For the controls, peripheral fasting blood was drawn from the vein in the Study population morning. Plasma blood was centrifuged at 1500 r/min for From September 2017 to January 2018, we consecutively a period of 10 min at 20°C, and the supernatant was recruited Chinese patients with CAD aged between 30 collected. Serum blood was centrifuged at 1000 r/min for a and 100 years who were hospitalized for coronary period of 10 min at 20°C, and the supernatant was angiography in the Peking Union Medical College collected. They were collected into sodium heparin tubes Hospital. Patients with the presence of ≥50% stenosis (BD) and stored at 80°C within 4 h until RIP3 levels were in ≥1 main coronary artery were diagnosed as having tested. Plasma/serum RIP3 levels were detected with ELISA CAD. Patients with CAD were further divided into three kits (Cusabio, Wuhan, China) according to the manufac- subgroups as follows: (1) patients with stable coronary turer’s instructions. artery disease (SCAD); (2) patients with unstable angina (UA); and (3) patients with MI. They were also Other parameters categorized according to the number of pathological branches involved. Chinese volunteers free from a known Subjects’ body mass index was calculated as weight (kg) history of CAD (as controls) were recruited from the divided by height squared (m ). Blood pressure was Peking Union Medical College Hospital at the same time. measured with a standard mercury sphygmomanometer by Subjects with acute renal injury, sepsis, or shock were specially trained nurses with the patient in a seated position excluded from the study. after ≥5 min of rest. Hypertension and diabetes were [13,14] diagnosed using the current guidelines. Peripheral UA was defined as a normal measurement of cardiac venous blood was taken from the antecubital vein early in troponin and with at least one of the following criteria: the morning while the patient was in a fasting state and prolonged (>20 min) angina pain at rest, new onset angina before any medications were administered. The levels of (Class II or III according to the Classification of the each lipid profile component (total cholesterol, low-density Canadian Cardiovascular Society), recent destabilization lipoprotein cholesterol, high-density lipoprotein cholester- of previously stable angina with at least Canadian ol, and triglycerides), creatinine, and high-sensitivity C Cardiovascular Society Class III angina characteristics reactive protein were measured. [9] (crescendo angina), or post-MI angina. SCAD was defined as angina that did not fulfill the above UA Statistical analysis [10] criteria. MI was defined as a rise and/or fall of cardiac troponin with at least one value above the 99th percentile The Shapiro-Wilk test was used to determine normality of upper reference limit and with at least one of the following: the data distribution for sample n < 50. The Kolmogorov- (1) ischemia symptoms; (2) new or presumed new Smirnov test was used to determine normality of the data significant ST-segment-T wave changes or new left bundle distribution for sample n ≥ 50. Normally distributed branch block; (3) development of pathological Q waves in data were expressed as the mean ± standard deviation, the electrocardiogram; (4) imaging evidence of new viable and data with non-normal distributions were expressed as myocardium loss or new regional wall motion abnormali- the median (Q1, Q3). Continuous, normally distributed ty; and (5) identification of an intra-coronary thrombus by variables between two groups were analyzed by Student t- [11] angiography or autopsy. 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 Coronary angiography and image interpretation analyzed by a one-way analysis of variance. The Kruskal- Coronary angiography was performed with a convention- Wallis H test was applied for data of this type that were not al angiography unit (Integris H; Philips Medical Systems, normally distributed. Categorical variables were summa- Amsterdam, the Netherlands). Coronary artery stenosis rized as count and percentage and were compared by the was imaged in the center of the field from multiple Chi-square test. Logistic regression was used to determine 1401 Chinese Medical Journal 2019;132(12) www.cmj.org the relationship between plasma RIP3 levels and CAD after Differences between plasma and serum RIP3 levels adjusting for confounding factors. The Youden’s index in Both plasma and serum levels of RIP3 were tested in 23 the receiver operating characteristic (ROC) curves was controls, 20 patients with SCAD, 20 patients with UA, and used to determine the optimal cut-off of plasma RIP3 levels 20 patients with MI. The median levels of plasma RIP3 for detecting CAD. Plasma RIP3 levels were compared were significantly higher than corresponding serum levels among controls and patients in different subtypes of CAD in total (433.75 [296.28, 516.11] pg/mL vs. 39.20 [20.43, using the analysis of variance type of trend analysis. To 65.33] pg/mL, Z = 257.000, P < 0.001) and in each group explore the relationship between plasma RIP3 levels and (controls: 244.28 [171.34, 287.03] pg/mL vs. 24.37 proportion of cases of ACS in subjects, all subjects were [16.26, 39.44] pg/mL, Z = 0.000, P < 0.001; patients divided into four categories according to the quartiles of with SCAD: 426.98 [395.70, 493.35] pg/mL vs. 34.67 plasma RIP3 levels. The correlation between plasma RIP3 [22.00, 51.31] pg/mL, Z = 0.000, P < 0.001; patients with and GSS was calculated using multiple linear regression UA: 464.98 [455.20, 512.51] pg/mL vs. 30.21 [16.08, models and adjusted for confounding factors. A P < 0.05 61.10] pg/mL, Z = 0.000, P < 0.001; patients with MI: was considered statistically significant. Statistical analysis 551.34 [473.64, 656.40] pg/mL vs. 105.23 [59.68, was performed using SPSS Statistics software, version 22.0 178.03] pg/mL, Z = 0.000, P < 0.001) [Figure 1]. (SPSS Inc., Chicago, IL, USA). Results Biomarker for CAD and plasma RIP3 cut-off level We tried to determine if plasma levels of RIP3 could Participant characteristics potentially serve as a marker for CAD. We expanded A total of 484 subjects were enrolled in this study, the sample size of RIP3 plasma level testing to include all including 166 controls and 318 patients with CAD 318 patients with CAD and 166 controls. The median (patients with SCAD, n = 93; patients with UA, n = 153; levels of plasma RIP3 in patients with CAD were and patients with MI, n = 72). The mean ages were 60.1  significantly higher than that of controls (406.87 [311.51, ± 9.4 years for controls and 64.4 ± 10.8 years for patients 516.59] pg/mL vs. 241.61 [175.83, 318.13] pg/mL, with CAD. Traditional cardiovascular risk factors and Z = 9565.000, P < 0.001). To diminish the confounding New York Heart Association classes in the study effects of traditional CAD risk factors, we conducted a population are summarized in Table 1. Most patients logistic regression analysis. The Youden’s index in with CAD were male and more likely to have a history of the ROC curve was used to determine the optimal cut-off hypertension and diabetes. CAD groups exhibited rela- of plasma RIP3 levels for detecting CAD. According to tively higher high-sensitivity C-reactive protein levels, the ROC curve analysis, the optimal cut-off value for compared with the controls. plasma RIP3 levels in predicting CAD was found to be Table 1: Baseline characteristics of study participants. Cardiovascular Controls SCAD patients UA patients MI patients Statistical risk factors (n = 166) (n = 93) (n = 153) (n = 72) values P x,jj Age (years) 60.1 ± 9.4 66.2 ± 8.8 63.7 ± 11.4 63.3 ± 11.7 7.770 <0.001 Male 106 (63.9) 61 (65.6) 102 (66.7) 56 (77.8) 4.622 0.202 2 jj BMI (kg/m ) 24.8 ± 3.2 25.3 ± 3.4 25.7 ± 3.6 25.5 ± 3.5 1.667 0.171 † x,jj,¶ HTN 41 (24.7) 68 (73.1) 103 (67.3) 49 (68.1) 87.693 <0.001 † x,jj,¶ DM 10 (6.0) 39 (41.9) 64 (41.8) 25 (34.7) 64.083 <0.001 † jj,¶ Currently smoking 44 (28.0) 38 (40.9) 78 (51.0) 44 (61.1) 28.156 <0.001 CAD family history 35 (22.3) 29 (31.2) 39 (25.5) 21 (29.2) 2.806 0.423 † x,jj,¶ NYHA Class 113.281 <0.001 I 166 (100.0) 53 (57.0) 77 (50.3) 47 (65.3) II 0 32 (34.4) 59 (38.6) 16 (22.2) III 0 7 (7.5) 15 (9.8) 7 (9.7) IV 0 1 (1.1) 2 (1.3) 2 (2.8) ∗∗ ‡ x,jj,¶, TC (mmol/L) 4.65 (4.08, 5.15) 3.78 (3.20, 4.35) 3.72 (3.24, 4.29) 4.04 (3.60, 4.90) 64.598 <0.001 ‡ jj,¶ TG (mmol/L) 1.13 (0.88, 1.49) 1.20 (0.91, 1.72) 1.33 (0.97, 1.90) 1.35 (0.96, 2.12) 18.470 <0.001 ∗∗ ‡ x,jj, LDL-C (mmol/L) 2.83 (2.32, 3.20) 2.06 (1.64, 2.55) 2.16 (1.72, 2.67) 2.41 (1.98, 3.18) 60.012 <0.001 ‡ x,jj,¶,‡‡ HDL-C (mmol/L) 1.20 (1.05, 1.37) 1.04 (0.85, 1.21) 0.94 (0.80, 1.08) 0.96 (0.83, 1.09) 94.815 <0.001 ‡ jj,¶ Creatinine (mmol/L) 72.00 (60.75, 80.00) 74.00 (67.00, 85.00) 80.00 (70.00, 93.00) 80.00 (68.25, 96.00) 31.790 <0.001 ∗∗ ‡ jj,¶, ,†† hs-CRP (mg/L) 0.75 (0.45, 2.15) 1.23 (0.52, 3.09) 1.54 (0.69, 3.84) 3.44 (1.23, 13.37) 38.472 <0.001 † 2 ‡ x Data were shown as mean ± standard deviation, median (Q1, Q3), or n (%). F values; x values; Z values; Comparison between controls and patients jj ¶ with SCAD, P < 0.010; Comparison between controls and patients with UA, P < 0.010; Comparison between controls and patients with MI, ∗∗ ‡‡ P < 0.010; Comparison between patients with SCAD and UA, P < 0.010; Comparison between patients with SCAD and MI, P < 0.010; †† Comparison between patients with UA and MI, P < 0.010. BMI: Body mass index; CAD: Coronary artery disease; DM: Diabetic mellitus; HDL-C: High-density lipoprotein cholesterol; hs-CRP: High-sensitive C-reactive protein; HTN: Hypertension; LDL-C: Low-density lipoprotein cholesterol; MI: Myocardial infarction; NYHA: New York Heart Association; SCAD: Stable coronary artery disease; TC: Total cholesterol; TG: Triglycerides; UA: Unstable angina. 1402 Chinese Medical Journal 2019;132(12) www.cmj.org 324.51 pg/mL, with a specificity of 80.0% and sensitivity of 500.04] pg/mL), and triple vessel disease (401.26 [311.09, 73.0% (area under curve = 0.819, 95% confidence inter- 521.91] pg/mL) [Supplemental Figure 1, http://links.lww. val = 0.779–0.859) [Figure 2]. After adjusting for con- com/CM9/A34]. However, there were significant differ- founding factors, plasma RIP3 levels still remained strongly ences in plasma RIP3 levels between the subjects with associated with CAD (P < 0.001) [Table 2]. normal coronary arteries and patients with different pathological branches involved. Association between plasma RIP3 levels and CAD severity To explore the relationship between plasma RIP3 levels Plasma RIP3 levels increased linearly from controls and proportion of cases of ACS in subjects, all subjects (241.61 [175.83, 318.13] pg/mL), to patients with SCAD were divided into four categories according to which (388.39 [328.8, 501.66] pg/mL), then patients with UA quartile their plasma RIP3 level fell into. Clinical (386.91 [303.14, 478.67] pg/mL), and finally to patients characteristics are listed in Supplementary Table 1, with MI (455.04 [343.36, 607.44] pg/mL; P for linear http://links.lww.com/CM9/A34. Subjects with higher trend < 0.001) [Figure 3]. We did not find a similar linear plasma RIP3 levels were older and were more likely to relationship in plasma RIP3 levels when comparing suffer from comorbidities. Moreover, they had higher patients with normal coronary arteries (245.24 [177.47, levels of high-sensitivity C-reactive protein. Notably, we 319.58] pg/mL), single vessel disease (403.53 [311.99, 505.46] pg/mL), double vessel disease (416.56 [297.74, Table 2: Logistic regression analyses of plasma RIP3 levels in diagnosing the occurrence of CAD. Items OR 95% CI P Model 1 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 11.98 7.30–19.65 <0.001 Model 2 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 10.67 6.18–18.43 <0.001 Model 3 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 6.00 3.04–11.81 <0.001 Model 1: adjusted for age, gender, smoking status, and CAD family history. Model 2: adjusted for age, gender, smoking status, CAD family Figure 1: Plasma and serum levels of RIP3 in controls, patients with SCAD, patients with history, body mass index, hypertension, diabetes. Model 3: adjusted for UA, and patients with MI. Short horizontal lines denote minimum value and maximum value; age, gender, smoking status, CAD family history, body mass index, long horizontal lines denote 25th, 50th, and 75th percentiles. P < 0.001, plasma levels vs. hypertension, diabetes, total cholesterol, low-density lipoprotein choles- serum levels (Mann-Whitney U test was used). MI: Myocardial infarction; RIP3: Receptor terol, high-density lipoprotein cholesterol, triglycerides and high-sensitive interacting protein kinase-3; SCAD: Stable coronary artery disease; UA: Unstable angina. C reactive protein. The optimal cut-off value for plasma RIP3 levels in predicting CAD was found to be 324.51 pg/mL. CAD: Coronary artery disease; CI: Confidence interval; OR: Odds ratio; RIP3: Receptor interacting protein kinase-3. Figure 3: Plasma RIP3 levels in controls and different subtypes of CAD. Short horizontal lines denote minimum value and maximum value; long horizontal lines denote 25th, 50th, and 75th percentiles. The analysis of variance type of trend analysis was used. CAD: Figure 2: Receiver operating characteristic curve to detect CAD. The optimal cut-off value Coronary artery disease; MI: Myocardial infarction; RIP3: Receptor interacting protein for plasma RIP3 levels in predicting CAD was found to be 324.51 pg/mL. CAD: Coronary kinase-3; SCAD: Stable coronary artery disease; UA: Unstable angina. artery disease; RIP3: Receptor interacting protein kinase-3. 1403 Chinese Medical Journal 2019;132(12) www.cmj.org found a significant positive correlation between proportion It is notable that we demonstrated a significant association of cases of ACS in subjects and the plasma RIP3 level between plasma RIP3 level quartiles and prevalence of quartile [Supplementary Figure 2, http://links.lww.com/ ACS cases, which might shed light in predicting adverse [24] CM9/A34]. events in patients with CAD. In 1989, Muller et al proposed the concept of “vulnerable plaque.” Predomi- We adopted multiple linear regression models to determine nantly derived from pathological observations at autop- the correlation between plasma RIP3 levels and GSS. sies, the “plaque-rupture” hypothesis and new views of Plasma RIP3 levels were associated with GSS after vulnerable atherosclerotic coronary plaque features were [25] adjustments for traditional cardiovascular risk factors established. Acute coronary thrombosis and subsequent (P < 0.05) [Table 3]. acute MI are now regarded as the results of sudden plaque [26,27] rupture or erosion. And efforts have been made to identify high-risk patients using advanced imaging meth- ods to find “vulnerable” coronary sites. However, these Discussion methods have many disadvantages, such as price, [28] This study confirmed that in patients with CAD plasma inconvenience, and poor efficacy. Karunakaran [23] RIP3 levels were significantly higher than controls and they et al found that necroptotic cell death in humans was were strongly associated with CAD, even after adjusting activated in advanced atherosclerotic plaques and for confounding factors. We believed that plasma RIP3 is expression of RIP3 and mixed lineage kinase domain- an independent CAD risk factor and might be regarded as like protein was increased with unstable carotid a surrogate marker for CAD screening. Plasma RIP3 levels atherosclerosis. This indicated that RIP3 might correlate were also found to be positively correlated with coronary with lesion vulnerability. The elevated plasma RIP3 levels arterial atherosclerosis severity and its clinical manifes- we found in patients with MI might be due to the rupture tations. This indicated that plasma RIP3 could be used to of atherosclerotic plaques with their subsequent release predict disease severity. into circulating plasma. This finding might also be related to thrombus formations in the coronary artery. [15] [29] The human RIP3 gene is located on chromosome 14, Zhang et al demonstrated a role for RIP3 in and its messenger RNA encodes a polypeptide of 518 promoting in vivo thrombosis and hemostasis. RIP3 amino acids. Several studies have pointed out that RIP3 is inhibitors dose-dependently inhibited platelet aggrega- essential for the execution of tumor necrosis factor- tion in vitro and prevented arterial thrombus formation [16-18] [29] induced necroptosis downstream of RIP1. In addi- in vivo. tion, through the analysis of RIP3 deficient mice, researchers have implicated the role of necroptosis in In this study, we found that plasma levels of RIP3 were [19] [20] atherosclerosis, alcoholic liver disease, and retinal significantly higher than serum levels of RIP3 in human [21,22] degeneration. The critical pro-necroptotic factor patients. Circulating RIP3 levels have been considered RIP3 is found to be present and activated in human important markers in diagnosing and predicting diseases. [23] [8] atherosclerotic plaque. RIP3 deficient, atherosclerosis- Kashlov et al figured out that levels of serum RIP3 were prone low-density lipoprotein receptor-deficient, or apoli- not altered significantly in patients with ST-segment poprotein E-deficient mice had a significant reduction in elevation MI, compared to controls. However, in our advanced atherosclerotic lesions. In addition, in vitro study, we pointed out that plasma levels of RIP3 were cellular studies showed that RIP3 deletion prevented significantly higher than the corresponding serum levels. [19] [29] primary necrosis of macrophages. These findings Zhang et al confirmed that RIP3 was expressed in indicated a driver function of RIP3 in atherosclerotic platelets, and the differences between serum and plasma necrosis. In the present study, plasma RIP3 levels were values might be influenced by the platelets at the time of associated with GSS. However, the extent to which blood coagulation. necroptosis and its mediator RIP3 play a role in atherosclerosis is unknown. 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. Table 3: Regression analysis of factors associated with Gensini Therefore, further studies, utilizing a prospective cohort, score. are needed to confirm the association between plasma RIP3 levels and CAD. The underlying mechanism behind Variables B Standard error P our findings remains uncertain, since the source of RIP3 Plasma RIP3 0.033 0.011 0.002 detected in plasma is still unclear. Additionally, partic- Plasma RIP3 0.031 0.011 0.004 ipants in our study came from China; thus, generalizability Plasma RIP3 0.027 0.012 0.020 to other ethnic or racial populations may not be valid. Similar research is necessary in other regions to assess the Adjusted for age, gender, smoking status, and family history. Adjusted general application of our findings. for age, gender, smoking status, family history, body mass index, hypertension, diabetes, acute coronary syndrome, total cholesterol, low- density lipoprotein cholesterol, high-density lipoprotein cholesterol and In conclusion, the results of this study confirmed that triglycerides. Adjusted for age, gender, smoking status, family history, compared to serum, plasma RIP3 levels were detected in body mass index, hypertension, diabetes, acute coronary syndrome, total significantly higher concentrations. Plasma RIP3 could be cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein used as a surrogate marker for CAD screening or a cholesterol, triglycerides, creatinine and high-sensitivity C-reactive protein. RIP3: Receptor interacting protein kinase-3. predictor for the severity of CAD progression. Further- 1404 Chinese Medical Journal 2019;132(12) www.cmj.org 14. Whelton PK, Carey RM, Aronow WS, Casey DE Jr, Collins KJ, more, plasma RIP3 levels were positively associated with Dennison Himmelfarb C, et al. 2017 ACC/AHA/AAPA/ABC/ CAD subtypes and proportion of cases of ACS in subjects. ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the pre- Accordingly, elevated plasma RIP3 levels may indicate the vention, detection, evaluation, and management of high blood need for a careful evaluation for the presence and pressure in adults: a report of the American College of Cardiology/ progression of CAD. American Heart Association Task Force on clinical practice guide- lines. 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Chin Med J 10.1016/s0002-9149(83)80105-2. 2019;132:1400–1405. doi: 10.1097/CM9.0000000000000225 13. American Diabetes A. 2. Classification and diagnosis of diabetes: standards of medical care in diabetes-2018. Diabetes Care 2018;41: S13–S27. doi: 10.2337/dc18-S002. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Chinese Medical Journal Wolters Kluwer Health

Plasma levels of receptor interacting protein kinase-3 correlated with coronary artery disease

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Abstract

Background: Necroptosis plays an important role in human atherosclerosis and atheroma development. Since receptor interacting protein kinase-3 (RIP3) acts as a key mediator of necroptosis, this study aimed to explore its relationship between plasma RIP3 levels and coronary artery disease (CAD) and discover a potential new biomarker for screening CAD subtypes and severity. Methods: A total of 318 patients with CAD who had coronary angiography and 166 controls in Peking Union Medical College Hospital from September 2017 to January 2018 were enrolled in this study. Patients with CAD were divided into three subgroups: patients with stable coronary artery disease (SCAD), patients with unstable angina (UA), and patients with myocardial infarction (MI). The severity of atherosclerosis was determined by Gensini score (GSS). Logistic regression was used to determine the relationship between plasma RIP3 levels and CAD. The correlation between plasma RIP3 and GSS was calculated using multiple linear regression models. Results: Overall, plasma RIP3 levels were significantly higher than serum RIP3 levels. Plasma RIP3 levels in patients with CAD were significantly higher than those in controls. Plasma RIP3 levels were strongly associated with CAD (odds ratio: 6.00, 95% confidence interval 3.04–11.81; P < 0.001). Plasma RIP3 levels increased linearly from controls to patients with SCAD, then patients with UA, and finally to patients with MI. We found a significantly positive correlation between proportion of cases of acute coronary syndrome in subjects and their plasma RIP3 level quartile. Plasma RIP3 levels were also associated with GSS (B 0.027; standard error 0.012; P < 0.05). Conclusions: Plasma RIP3 levels were independently associated with CAD. Plasma RIP3 levels could potentially supplement clinical assessment to screen CAD and determine CAD severity. Keywords: Receptor interacting protein kinase-3; Coronary artery disease; Necroptosis; Atherosclerosis; Acute coronary syndrome; Biomarker Introduction Emerging pieces of evidence have suggested that necrop- tosis might play an important role in several inflammatory Coronary artery disease (CAD) is common in developed conditions and diseases, including human atherosclerosis [1,2] countries and remains the leading cause of death. A [4] and atheroma development. Receptor interacting pro- study has focused on the pathogenesis of atherosclerosis tein kinase-3 (RIP3) is currently regarded as a critical [3] and the development of CAD. However, several [5] regulator of programmed necrosis/necroptosis. As far as questions remain unanswered. First, it would be better if we know, circulating RIP3 levels have been considered new biomarkers could be found to assist in screening CAD important markers in diagnosing and predicting some subtypes based on current definitions. Second, we need [6] [7] diseases, including sepsis, acute kidney injury, and ST- more convenient and non-invasive methods to evaluate the [8] segment elevation myocardial infarction (MI). It was coronary artery atheromatous severity instead of coronary reported that the levels of serum RIP3 were not altered computed tomography angiography and coronary arteri- significantly in patients with ST-segment elevation MI ography. Moreover, it is hard but important to select [8] compared to controls. However, the association between patients who might be suffering with acute coronary RIP3 levels and CAD required further confirmation in a syndrome (ACS) from others so that appropriate and timely prevention and treatment can be given. Xiao-Min Hu and Xi Chen contributed equally to this study. Access this article online Correspondence to: Prof. Shu-Yang Zhang, Department of Cardiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Quick Response Code: Medical College, No.1 Shuaifuyuan, Dongcheng District, Beijing 100730, China Website: E-Mail: shuyangzhang103@163.com www.cmj.org Copyright © 2019 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license. This is an open access article distributed under the terms of the Creative DOI: Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is 10.1097/CM9.0000000000000225 permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Chinese Medical Journal 2019;132(12) Received: 07-01-2019 Edited by: Xin Chen Downloaded from http://journals.lww.com/cmj by BhDMf5ePHKbH4TTImqenVA5KvPVPZ0P5BEgU+IUTEfzO/GUWifn2IfwcEVVH9SSn on 06/01/2020 Chinese Medical Journal 2019;132(12) www.cmj.org large-scale study. And the differences between plasma projections. Using the quantitative coronary analysis RIP3 levels and serum RIP3 levels have not been discussed. system, the coronary atherosclerotic lesion severity was evaluated from at least two projections by two interven- The aim of this study was to confirm the relationship tional cardiologists with at least 5 years of experience who between plasma RIP3 levels and CAD. Furthermore, we were blinded to the patients’ clinical information. Coro- tried to explore the relationship between plasma RIP3 nary angiographies were performed to determine the [12] levels and various CAD subtypes and severity. severity of atherosclerosis by the Gensini score (GSS). Methods Plasma/serum collection and plasma/serum RIP3 level quantification Ethical approval For the patients with MI, peripheral blood was whether This study was approved by the Ethics Committee of drawn from the radial or femoral artery before coronary Peking Union Medical College Hospital and conducted angiography or from the vein in the morning on the day according to the principles of the Declaration of Helsinki. after admission according to their disease subtypes and Written informed consent was obtained from all treatment guidelines. For the patients with SCAD and UA, participants. peripheral fasting blood was drawn from the vein in the morning the day after admission. For the controls, peripheral fasting blood was drawn from the vein in the Study population morning. Plasma blood was centrifuged at 1500 r/min for From September 2017 to January 2018, we consecutively a period of 10 min at 20°C, and the supernatant was recruited Chinese patients with CAD aged between 30 collected. Serum blood was centrifuged at 1000 r/min for a and 100 years who were hospitalized for coronary period of 10 min at 20°C, and the supernatant was angiography in the Peking Union Medical College collected. They were collected into sodium heparin tubes Hospital. Patients with the presence of ≥50% stenosis (BD) and stored at 80°C within 4 h until RIP3 levels were in ≥1 main coronary artery were diagnosed as having tested. Plasma/serum RIP3 levels were detected with ELISA CAD. Patients with CAD were further divided into three kits (Cusabio, Wuhan, China) according to the manufac- subgroups as follows: (1) patients with stable coronary turer’s instructions. artery disease (SCAD); (2) patients with unstable angina (UA); and (3) patients with MI. They were also Other parameters categorized according to the number of pathological branches involved. Chinese volunteers free from a known Subjects’ body mass index was calculated as weight (kg) history of CAD (as controls) were recruited from the divided by height squared (m ). Blood pressure was Peking Union Medical College Hospital at the same time. measured with a standard mercury sphygmomanometer by Subjects with acute renal injury, sepsis, or shock were specially trained nurses with the patient in a seated position excluded from the study. after ≥5 min of rest. Hypertension and diabetes were [13,14] diagnosed using the current guidelines. Peripheral UA was defined as a normal measurement of cardiac venous blood was taken from the antecubital vein early in troponin and with at least one of the following criteria: the morning while the patient was in a fasting state and prolonged (>20 min) angina pain at rest, new onset angina before any medications were administered. The levels of (Class II or III according to the Classification of the each lipid profile component (total cholesterol, low-density Canadian Cardiovascular Society), recent destabilization lipoprotein cholesterol, high-density lipoprotein cholester- of previously stable angina with at least Canadian ol, and triglycerides), creatinine, and high-sensitivity C Cardiovascular Society Class III angina characteristics reactive protein were measured. [9] (crescendo angina), or post-MI angina. SCAD was defined as angina that did not fulfill the above UA Statistical analysis [10] criteria. MI was defined as a rise and/or fall of cardiac troponin with at least one value above the 99th percentile The Shapiro-Wilk test was used to determine normality of upper reference limit and with at least one of the following: the data distribution for sample n < 50. The Kolmogorov- (1) ischemia symptoms; (2) new or presumed new Smirnov test was used to determine normality of the data significant ST-segment-T wave changes or new left bundle distribution for sample n ≥ 50. Normally distributed branch block; (3) development of pathological Q waves in data were expressed as the mean ± standard deviation, the electrocardiogram; (4) imaging evidence of new viable and data with non-normal distributions were expressed as myocardium loss or new regional wall motion abnormali- the median (Q1, Q3). Continuous, normally distributed ty; and (5) identification of an intra-coronary thrombus by variables between two groups were analyzed by Student t- [11] angiography or autopsy. 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 Coronary angiography and image interpretation analyzed by a one-way analysis of variance. The Kruskal- Coronary angiography was performed with a convention- Wallis H test was applied for data of this type that were not al angiography unit (Integris H; Philips Medical Systems, normally distributed. Categorical variables were summa- Amsterdam, the Netherlands). Coronary artery stenosis rized as count and percentage and were compared by the was imaged in the center of the field from multiple Chi-square test. Logistic regression was used to determine 1401 Chinese Medical Journal 2019;132(12) www.cmj.org the relationship between plasma RIP3 levels and CAD after Differences between plasma and serum RIP3 levels adjusting for confounding factors. The Youden’s index in Both plasma and serum levels of RIP3 were tested in 23 the receiver operating characteristic (ROC) curves was controls, 20 patients with SCAD, 20 patients with UA, and used to determine the optimal cut-off of plasma RIP3 levels 20 patients with MI. The median levels of plasma RIP3 for detecting CAD. Plasma RIP3 levels were compared were significantly higher than corresponding serum levels among controls and patients in different subtypes of CAD in total (433.75 [296.28, 516.11] pg/mL vs. 39.20 [20.43, using the analysis of variance type of trend analysis. To 65.33] pg/mL, Z = 257.000, P < 0.001) and in each group explore the relationship between plasma RIP3 levels and (controls: 244.28 [171.34, 287.03] pg/mL vs. 24.37 proportion of cases of ACS in subjects, all subjects were [16.26, 39.44] pg/mL, Z = 0.000, P < 0.001; patients divided into four categories according to the quartiles of with SCAD: 426.98 [395.70, 493.35] pg/mL vs. 34.67 plasma RIP3 levels. The correlation between plasma RIP3 [22.00, 51.31] pg/mL, Z = 0.000, P < 0.001; patients with and GSS was calculated using multiple linear regression UA: 464.98 [455.20, 512.51] pg/mL vs. 30.21 [16.08, models and adjusted for confounding factors. A P < 0.05 61.10] pg/mL, Z = 0.000, P < 0.001; patients with MI: was considered statistically significant. Statistical analysis 551.34 [473.64, 656.40] pg/mL vs. 105.23 [59.68, was performed using SPSS Statistics software, version 22.0 178.03] pg/mL, Z = 0.000, P < 0.001) [Figure 1]. (SPSS Inc., Chicago, IL, USA). Results Biomarker for CAD and plasma RIP3 cut-off level We tried to determine if plasma levels of RIP3 could Participant characteristics potentially serve as a marker for CAD. We expanded A total of 484 subjects were enrolled in this study, the sample size of RIP3 plasma level testing to include all including 166 controls and 318 patients with CAD 318 patients with CAD and 166 controls. The median (patients with SCAD, n = 93; patients with UA, n = 153; levels of plasma RIP3 in patients with CAD were and patients with MI, n = 72). The mean ages were 60.1  significantly higher than that of controls (406.87 [311.51, ± 9.4 years for controls and 64.4 ± 10.8 years for patients 516.59] pg/mL vs. 241.61 [175.83, 318.13] pg/mL, with CAD. Traditional cardiovascular risk factors and Z = 9565.000, P < 0.001). To diminish the confounding New York Heart Association classes in the study effects of traditional CAD risk factors, we conducted a population are summarized in Table 1. Most patients logistic regression analysis. The Youden’s index in with CAD were male and more likely to have a history of the ROC curve was used to determine the optimal cut-off hypertension and diabetes. CAD groups exhibited rela- of plasma RIP3 levels for detecting CAD. According to tively higher high-sensitivity C-reactive protein levels, the ROC curve analysis, the optimal cut-off value for compared with the controls. plasma RIP3 levels in predicting CAD was found to be Table 1: Baseline characteristics of study participants. Cardiovascular Controls SCAD patients UA patients MI patients Statistical risk factors (n = 166) (n = 93) (n = 153) (n = 72) values P x,jj Age (years) 60.1 ± 9.4 66.2 ± 8.8 63.7 ± 11.4 63.3 ± 11.7 7.770 <0.001 Male 106 (63.9) 61 (65.6) 102 (66.7) 56 (77.8) 4.622 0.202 2 jj BMI (kg/m ) 24.8 ± 3.2 25.3 ± 3.4 25.7 ± 3.6 25.5 ± 3.5 1.667 0.171 † x,jj,¶ HTN 41 (24.7) 68 (73.1) 103 (67.3) 49 (68.1) 87.693 <0.001 † x,jj,¶ DM 10 (6.0) 39 (41.9) 64 (41.8) 25 (34.7) 64.083 <0.001 † jj,¶ Currently smoking 44 (28.0) 38 (40.9) 78 (51.0) 44 (61.1) 28.156 <0.001 CAD family history 35 (22.3) 29 (31.2) 39 (25.5) 21 (29.2) 2.806 0.423 † x,jj,¶ NYHA Class 113.281 <0.001 I 166 (100.0) 53 (57.0) 77 (50.3) 47 (65.3) II 0 32 (34.4) 59 (38.6) 16 (22.2) III 0 7 (7.5) 15 (9.8) 7 (9.7) IV 0 1 (1.1) 2 (1.3) 2 (2.8) ∗∗ ‡ x,jj,¶, TC (mmol/L) 4.65 (4.08, 5.15) 3.78 (3.20, 4.35) 3.72 (3.24, 4.29) 4.04 (3.60, 4.90) 64.598 <0.001 ‡ jj,¶ TG (mmol/L) 1.13 (0.88, 1.49) 1.20 (0.91, 1.72) 1.33 (0.97, 1.90) 1.35 (0.96, 2.12) 18.470 <0.001 ∗∗ ‡ x,jj, LDL-C (mmol/L) 2.83 (2.32, 3.20) 2.06 (1.64, 2.55) 2.16 (1.72, 2.67) 2.41 (1.98, 3.18) 60.012 <0.001 ‡ x,jj,¶,‡‡ HDL-C (mmol/L) 1.20 (1.05, 1.37) 1.04 (0.85, 1.21) 0.94 (0.80, 1.08) 0.96 (0.83, 1.09) 94.815 <0.001 ‡ jj,¶ Creatinine (mmol/L) 72.00 (60.75, 80.00) 74.00 (67.00, 85.00) 80.00 (70.00, 93.00) 80.00 (68.25, 96.00) 31.790 <0.001 ∗∗ ‡ jj,¶, ,†† hs-CRP (mg/L) 0.75 (0.45, 2.15) 1.23 (0.52, 3.09) 1.54 (0.69, 3.84) 3.44 (1.23, 13.37) 38.472 <0.001 † 2 ‡ x Data were shown as mean ± standard deviation, median (Q1, Q3), or n (%). F values; x values; Z values; Comparison between controls and patients jj ¶ with SCAD, P < 0.010; Comparison between controls and patients with UA, P < 0.010; Comparison between controls and patients with MI, ∗∗ ‡‡ P < 0.010; Comparison between patients with SCAD and UA, P < 0.010; Comparison between patients with SCAD and MI, P < 0.010; †† Comparison between patients with UA and MI, P < 0.010. BMI: Body mass index; CAD: Coronary artery disease; DM: Diabetic mellitus; HDL-C: High-density lipoprotein cholesterol; hs-CRP: High-sensitive C-reactive protein; HTN: Hypertension; LDL-C: Low-density lipoprotein cholesterol; MI: Myocardial infarction; NYHA: New York Heart Association; SCAD: Stable coronary artery disease; TC: Total cholesterol; TG: Triglycerides; UA: Unstable angina. 1402 Chinese Medical Journal 2019;132(12) www.cmj.org 324.51 pg/mL, with a specificity of 80.0% and sensitivity of 500.04] pg/mL), and triple vessel disease (401.26 [311.09, 73.0% (area under curve = 0.819, 95% confidence inter- 521.91] pg/mL) [Supplemental Figure 1, http://links.lww. val = 0.779–0.859) [Figure 2]. After adjusting for con- com/CM9/A34]. However, there were significant differ- founding factors, plasma RIP3 levels still remained strongly ences in plasma RIP3 levels between the subjects with associated with CAD (P < 0.001) [Table 2]. normal coronary arteries and patients with different pathological branches involved. Association between plasma RIP3 levels and CAD severity To explore the relationship between plasma RIP3 levels Plasma RIP3 levels increased linearly from controls and proportion of cases of ACS in subjects, all subjects (241.61 [175.83, 318.13] pg/mL), to patients with SCAD were divided into four categories according to which (388.39 [328.8, 501.66] pg/mL), then patients with UA quartile their plasma RIP3 level fell into. Clinical (386.91 [303.14, 478.67] pg/mL), and finally to patients characteristics are listed in Supplementary Table 1, with MI (455.04 [343.36, 607.44] pg/mL; P for linear http://links.lww.com/CM9/A34. Subjects with higher trend < 0.001) [Figure 3]. We did not find a similar linear plasma RIP3 levels were older and were more likely to relationship in plasma RIP3 levels when comparing suffer from comorbidities. Moreover, they had higher patients with normal coronary arteries (245.24 [177.47, levels of high-sensitivity C-reactive protein. Notably, we 319.58] pg/mL), single vessel disease (403.53 [311.99, 505.46] pg/mL), double vessel disease (416.56 [297.74, Table 2: Logistic regression analyses of plasma RIP3 levels in diagnosing the occurrence of CAD. Items OR 95% CI P Model 1 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 11.98 7.30–19.65 <0.001 Model 2 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 10.67 6.18–18.43 <0.001 Model 3 Plasma RIP3 <324.51 pg/mL 1.00 –– Plasma RIP3 >324.51 pg/mL 6.00 3.04–11.81 <0.001 Model 1: adjusted for age, gender, smoking status, and CAD family history. Model 2: adjusted for age, gender, smoking status, CAD family Figure 1: Plasma and serum levels of RIP3 in controls, patients with SCAD, patients with history, body mass index, hypertension, diabetes. Model 3: adjusted for UA, and patients with MI. Short horizontal lines denote minimum value and maximum value; age, gender, smoking status, CAD family history, body mass index, long horizontal lines denote 25th, 50th, and 75th percentiles. P < 0.001, plasma levels vs. hypertension, diabetes, total cholesterol, low-density lipoprotein choles- serum levels (Mann-Whitney U test was used). MI: Myocardial infarction; RIP3: Receptor terol, high-density lipoprotein cholesterol, triglycerides and high-sensitive interacting protein kinase-3; SCAD: Stable coronary artery disease; UA: Unstable angina. C reactive protein. The optimal cut-off value for plasma RIP3 levels in predicting CAD was found to be 324.51 pg/mL. CAD: Coronary artery disease; CI: Confidence interval; OR: Odds ratio; RIP3: Receptor interacting protein kinase-3. Figure 3: Plasma RIP3 levels in controls and different subtypes of CAD. Short horizontal lines denote minimum value and maximum value; long horizontal lines denote 25th, 50th, and 75th percentiles. The analysis of variance type of trend analysis was used. CAD: Figure 2: Receiver operating characteristic curve to detect CAD. The optimal cut-off value Coronary artery disease; MI: Myocardial infarction; RIP3: Receptor interacting protein for plasma RIP3 levels in predicting CAD was found to be 324.51 pg/mL. CAD: Coronary kinase-3; SCAD: Stable coronary artery disease; UA: Unstable angina. artery disease; RIP3: Receptor interacting protein kinase-3. 1403 Chinese Medical Journal 2019;132(12) www.cmj.org found a significant positive correlation between proportion It is notable that we demonstrated a significant association of cases of ACS in subjects and the plasma RIP3 level between plasma RIP3 level quartiles and prevalence of quartile [Supplementary Figure 2, http://links.lww.com/ ACS cases, which might shed light in predicting adverse [24] CM9/A34]. events in patients with CAD. In 1989, Muller et al proposed the concept of “vulnerable plaque.” Predomi- We adopted multiple linear regression models to determine nantly derived from pathological observations at autop- the correlation between plasma RIP3 levels and GSS. sies, the “plaque-rupture” hypothesis and new views of Plasma RIP3 levels were associated with GSS after vulnerable atherosclerotic coronary plaque features were [25] adjustments for traditional cardiovascular risk factors established. Acute coronary thrombosis and subsequent (P < 0.05) [Table 3]. acute MI are now regarded as the results of sudden plaque [26,27] rupture or erosion. And efforts have been made to identify high-risk patients using advanced imaging meth- ods to find “vulnerable” coronary sites. However, these Discussion methods have many disadvantages, such as price, [28] This study confirmed that in patients with CAD plasma inconvenience, and poor efficacy. Karunakaran [23] RIP3 levels were significantly higher than controls and they et al found that necroptotic cell death in humans was were strongly associated with CAD, even after adjusting activated in advanced atherosclerotic plaques and for confounding factors. We believed that plasma RIP3 is expression of RIP3 and mixed lineage kinase domain- an independent CAD risk factor and might be regarded as like protein was increased with unstable carotid a surrogate marker for CAD screening. Plasma RIP3 levels atherosclerosis. This indicated that RIP3 might correlate were also found to be positively correlated with coronary with lesion vulnerability. The elevated plasma RIP3 levels arterial atherosclerosis severity and its clinical manifes- we found in patients with MI might be due to the rupture tations. This indicated that plasma RIP3 could be used to of atherosclerotic plaques with their subsequent release predict disease severity. into circulating plasma. This finding might also be related to thrombus formations in the coronary artery. [15] [29] The human RIP3 gene is located on chromosome 14, Zhang et al demonstrated a role for RIP3 in and its messenger RNA encodes a polypeptide of 518 promoting in vivo thrombosis and hemostasis. RIP3 amino acids. Several studies have pointed out that RIP3 is inhibitors dose-dependently inhibited platelet aggrega- essential for the execution of tumor necrosis factor- tion in vitro and prevented arterial thrombus formation [16-18] [29] induced necroptosis downstream of RIP1. In addi- in vivo. tion, through the analysis of RIP3 deficient mice, researchers have implicated the role of necroptosis in In this study, we found that plasma levels of RIP3 were [19] [20] atherosclerosis, alcoholic liver disease, and retinal significantly higher than serum levels of RIP3 in human [21,22] degeneration. The critical pro-necroptotic factor patients. Circulating RIP3 levels have been considered RIP3 is found to be present and activated in human important markers in diagnosing and predicting diseases. [23] [8] atherosclerotic plaque. RIP3 deficient, atherosclerosis- Kashlov et al figured out that levels of serum RIP3 were prone low-density lipoprotein receptor-deficient, or apoli- not altered significantly in patients with ST-segment poprotein E-deficient mice had a significant reduction in elevation MI, compared to controls. However, in our advanced atherosclerotic lesions. In addition, in vitro study, we pointed out that plasma levels of RIP3 were cellular studies showed that RIP3 deletion prevented significantly higher than the corresponding serum levels. [19] [29] primary necrosis of macrophages. These findings Zhang et al confirmed that RIP3 was expressed in indicated a driver function of RIP3 in atherosclerotic platelets, and the differences between serum and plasma necrosis. In the present study, plasma RIP3 levels were values might be influenced by the platelets at the time of associated with GSS. However, the extent to which blood coagulation. necroptosis and its mediator RIP3 play a role in atherosclerosis is unknown. 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. Table 3: Regression analysis of factors associated with Gensini Therefore, further studies, utilizing a prospective cohort, score. are needed to confirm the association between plasma RIP3 levels and CAD. The underlying mechanism behind Variables B Standard error P our findings remains uncertain, since the source of RIP3 Plasma RIP3 0.033 0.011 0.002 detected in plasma is still unclear. Additionally, partic- Plasma RIP3 0.031 0.011 0.004 ipants in our study came from China; thus, generalizability Plasma RIP3 0.027 0.012 0.020 to other ethnic or racial populations may not be valid. Similar research is necessary in other regions to assess the Adjusted for age, gender, smoking status, and family history. Adjusted general application of our findings. for age, gender, smoking status, family history, body mass index, hypertension, diabetes, acute coronary syndrome, total cholesterol, low- density lipoprotein cholesterol, high-density lipoprotein cholesterol and In conclusion, the results of this study confirmed that triglycerides. Adjusted for age, gender, smoking status, family history, compared to serum, plasma RIP3 levels were detected in body mass index, hypertension, diabetes, acute coronary syndrome, total significantly higher concentrations. 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Chinese Medical Journal – Wolters Kluwer Health

Published: Jun 1, 2019

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