Association between Eicosapentaenoic Acid to Arachidonic Acid Ratio and Characteristics of Plaque Rupture

Abstract

Aims: Eicosapentaenoic acid (EPA) has shown beneficial effects on coronary plaque stabilization. Based on our previous study, we speculated that EPA might be associated with the development of healed plaques and might limit thrombus size. This study aimed to elucidate the association between EPA and arachidonic acid (AA) ratios and various plaque characteristics in patients with plaque rupture.

Methods: A total of 95 patients with acute coronary syndrome (ACS) caused by plaque rupture who did not take lipid-lowering drugs and underwent percutaneous coronary intervention using optical coherence tomography (OCT) were included. Clinical characteristics, lipid profiles, and OCT findings were compared between patients with lower and higher EPA/AA ratios (0.41) according to the levels in the Japanese general population.

Results: In the high EPA/AA (n=29, 30.5%) and low EPA/AA (n=66, 69.5 %) groups, the high EPA/AA group was significantly older (76.1 vs. 66.1 years, P＜0.01) and had lower peak creatine kinase (556 vs. 1651 U/L, P=0.03) than those with low EPA/AA. Similarly, patients with high EPA/AA had higher prevalence of layered and calcified plaque (75.9 vs. 39.4 %, P＜0.01; 79.3 vs. 50.0 %, P＜0.01, respectively) than low EPA/AA group. Multivariate logistic regression analysis demonstrated that a high EPA/AA ratio was an independent factor in determining the development of layered and calcified plaques.

Conclusion: A high EPA/AA ratio may be associated with the development of layered and calcified plaques in patients with plaque rupture.

Introduction

Eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) with a broad range of potentially beneficial cardiovascular effects^1-5). Numerous epidemiological studies have shown that the long-term intake of n-3 PUFAs plays an important role in reducing the occurrence of cardiovascular events^6-8). In contrast, arachidonic acid (AA) is classified in the n-6 PUFAs category, and lower EPA/AA ratios are considered to be promising risk markers for coronary artery disease (CAD)^{9, 10)}. EPA prevented endothelial dysfunction^{11, 12)}, reduced platelet procoagulant activity¹³⁾, and stabilized atherosclerotic plaque composition through endothelial cells, smooth muscle cells and macrophages^{14, 15)}. Elevated balance of EPA/AA and various bioactive lipid mediators can promote anti-thrombotic and anti-inflammatory effects as well as anti-atherogenic lipid metabolism (Supplementary Fig.1)^16-18).

Supplementary Fig.1. The Impact of EPA and AA on plaque

EPA has been reported to have beneficial effects on multiple atherosclerosis processes including foam-cell formation, inflammation, plaque formation/progression, platelet aggregation, and thrombus formation.

EPA, eicosapentaenoic acid; AA, arachidonic acid.

Optical coherence tomography (OCT) is a high-resolution intracoronary imaging modality that allows the identification of atherosclerotic plaque characteristics, including lipid-rich plaques, thin-cap fibroatheromas, and plaque rupture¹⁹⁾. Several studies using OCT have been conducted on the impact of EPA/AA ratios on plaque characteristics^20-24). A study including 59 patients with acute coronary syndrome (ACS) reported that EPA/AA ratios were significantly lower in ACS patients with lipid-rich plaques, ruptured plaques, and thin-cap fibroatheromas, and positively correlated with fibrous cap thickness²⁰⁾. A recent multicenter randomized controlled trial evaluating 130 patients with ACS for non-culprit lesions using OCT revealed that a combination of EPA therapy (1.8 g/day) with statins significantly increased fibrous cap thickness in patients with thinner fibrous caps compared with therapy with a statin alone²²⁾. The association between a low EPA/AA ratio and plaque vulnerability, as well as the beneficial effect of EPA on plaque stabilization, has been demonstrated; however, it is not yet fully understood how the EPA/AA ratio affects plaque characteristics in patients with ACS caused by plaque rupture.

Plaque rupture, which is a common phenotype of ACS, consists of a large lipid core with a ruptured thin fibrous cap and an overlying thrombus^{25, 26)}. However, pathological studies have suggested that some plaques may rupture silently without causing symptoms, subsequently leading to healed plaques²⁷⁾. There is a predominance of thrombogenic resistance factors that inhibit thrombus formation and promote asymptomatic plaque healing²⁸⁾. A histological validation study demonstrated that OCT could demonstrate healed plaques as layered plaques with one or more layers of different optical densities²⁹⁾. In our study, we assessed the lipid profiles of 436 men with ACS who were not taking any lipid-lowering drugs and found that patients with thrombosis in myocardial infarction (TIMI) grade ≥ 1 had significantly higher EPA levels than those with TIMI grade 0 ³⁰⁾. We speculated that the higher EPA/AA ratios and EPA levels might be associated with the development of healed plaques and might limit the size of the thrombus overlying the coronary artery, which results in healing lesions and a delayed age of onset of ACS.

Aim

This study was designed to test our hypotheses. We investigated the association between the EPA/AA ratio and various plaque characteristics, such as layered plaques, in patients with ACS caused by plaque rupture using OCT. In accordance with our previous study, we set the EPA/AA ratio as 0.41 based on the median value of the Hisayama study³¹⁾, the general Japanese population study, and enrolled patients who were not taking any lipid-lowering medications.

Methods

Study Participants

A total of 95 patients with ACS caused by plaque rupture who did not take any lipid-lowering drugs, such as statins, ezetimibe, PCSK9 inhibitors, fibrates, or n-3 PUFA-containing drugs, before the onset of ACS were enrolled consecutively between April 2018 and December 2021 at Showa University Hospital. In this study, lesions with plaque erosion because of various pathologies, including lipidic plaque and spasm were excluded^32-34). Calcified nodules were also excluded because their pathogenesis is distinct from the lipid-associated pathogenesis of ACS.

Definitions of ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation ACS (NSTE-ACS), TIMI flow grade, extent of coronary vessel disease, body mass index, hypertension, diabetes mellitus, and dyslipidemia are shown in Supplement.

The Institutional Review Board of Showa University approved the study protocol (Nos. 2855 and 3045). This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Informed consent was obtained from all the participants.

Measurement of Biomarkers

Serum samples were collected immediately before emergency coronary angiography on admission for ACS. Plasma EPA and AA levels were measured by gas-liquid chromatography in a commercially available laboratory (BML Co., Ltd., Tokyo, Japan). Methods for measuring triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), glycated hemoglobin (HbA1c), low-density lipoprotein cholesterol (LDL-C), small dense LDL-C (sd-LDL-C), non-HDL-C, malondialdehyde-modified LDL (MDA-LDL), TG-rich lipoprotein cholesterol (TRL-C), lipoprotein (a), hs-CRP, N-terminal pro-brain natriuretic peptide (NT-proBNP), estimated glomerular filtration rate (eGFR), and peak creatine kinase (CK) are described in Supplement.

OCT Imaging Acquisition

After the completion of diagnostic coronary angiography, careful manual thrombectomy either using an aspiration catheter or through dilatation with a balloon measuring ≤ 2.0 mm when the TIMI flow remained 0 or 1 was performed, and the culprit lesions were observed using an OCT imaging catheter. However, if the TIMI flow was 2 or 3, the decision to perform these procedures before imaging with OCT was left to the operator. OCT images were acquired using a commercially available frequency-domain OCT imaging system (ILUMIEN; Abbott Vascular, St. Paul, MN, USA). Using this system, a 2.7-Fr OCT imaging catheter (Dragonfly™ OPTIS™; Abbott Vascular, St. Paul, MN, USA) was advanced distally to the lesion, and automated pullback was initiated in concordance with blood clearance upon injection of contrast media. All the images were de-identified and digitally stored.

OCT Analysis

All OCT images were analyzed using an offline review workstation. The sites selected for analysis were cross sections with the minimum lumen area and proximal and distal reference cross sections in the culprit lesion. The proximal and distal references were defined as the sites with the largest lumen diameter within 10 mm proximally and distally to the regions with the smallest lumen area and before any side branch³⁵⁾. The minimum lumen area was measured at the site with the smallest lumen area in the culprit lesion, whereas the reference lumen area was measured at the reference cross-section. Area stenosis was calculated as follows: [(mean reference lumen area−minimum lumen area)÷mean reference lumen area] ×100. Lesion length was defined as the region around the minimum lumen area where the lumen area was ＜50% of the largest reference lumen area³⁶⁾. The images were qualitatively and quantitatively analyzed at 0.2 mm intervals.

Plaque rupture was defined as a plaque with intimal tearing, disruption, or dissection of the cap¹⁹⁾. Thin-cap fibroatheroma was defined as a lipid-rich plaque (＞90°) overlaid with a thin fibrous cap (＜65 µm)³⁷⁾. Layered plaques were defined as plaques presenting in three or more consecutive frames, one or more layers with different optical densities, and a clear demarcation from the underlying components^{29, 38)}. Cholesterol crystal was defined as a thin, linear region of high-intensity^{19, 39)}. Macrophage accumulation was defined as a high-intensity signal-rich linear region with sharp attenuation. Macrophage signals were semi-quantitatively graded according to previous reports^{37, 40)}: grade 0, no macrophages; grade 1, localized macrophage accumulation; grade 2, clustered accumulation in ＜1 quadrant; grade 3, clustered accumulation in ＞1 quadrant but in ＜3 quadrants; and grade 4, clustered accumulation in ＞3 quadrants. To distinguish between grades 1 and 2, the degree of macrophage extension was defined as 30°. Microchannels were defined as small vesicular or tubular structures with diameters of 50–300 µm and differentiated from any other branch⁴¹⁾. The lipidic plaque was defined as a diffusely bordered, signal-poor region lipid pools⁴⁰⁾. The lipid arc was measured at 0.2-mm intervals throughout the length of each lesion, and the values were averaged. The length of the lipid was measured longitudinally. The lipid index was defined as the mean lipid arc multiplied by the lipid length⁴²⁾. Calcified plaque was identified as an area with a low backscattering signal and a sharp border¹⁹⁾. The calcification arc was measured at 0.2-mm intervals throughout the length of each lesion, and the values were averaged. The length of the calcification was also measured longitudinally. As with lipid index, the calcification index was defined as the mean calcification arc multiplied by the calcification length.

All OCT images were analyzed by two independent investigators (TS and TA) blinded to the angiographic and clinical findings, using an offline review workstation. When discordance in terms of qualitative plaque morphology occurred between observers, a consensus was reached with the assistance of a third investigator (HM).

Statistical Analysis

Statistical analyses were performed using JMP statistical software version 16 (SAS Institute, Cary, NC, USA). Patients were classified based on the EPA/AA ratio according to the general Japanese population, the Hisayama study³¹⁾.

Data are presented as mean±standard deviation, median with interquartile range, or number (proportion) as appropriate. Normality of distribution was determined using the Shapiro–Wilk test. Normally and non-normally distributed continuous variables were compared using the unpaired Student’s t-test and the Mann–Whitney U test or Wilcoxon signed-rank test, respectively. Categorical variables were compared using Fisher’s exact test or chi-square test. Multivariate logistic regression was used to determine risk factors for layered and calcified plaques. Multivariate regression analysis was used to determine significant factors associated with peak CK. Inter- and intra-observer reliabilities were assessed using kappa statistics. Thereafter, receiver operating characteristic (ROC) curves were constructed. The area under the curve (AUC), sensitivity, and specificity for predicting high EPA/AA ratio (≥ 0.41) were calculated, with an AUC of 0.50 indicating no accuracy and an AUC of 1.00 indicating maximum accuracy. All statistical analyses were two-tailed. Statistical significance was set at P＜0.05.

Results

Baseline Characteristics

Approximately 30% of patients (N=29) with ACS caused by plaque rupture were classified into the high EPA/AA group (Fig.1). The distribution of EPA and AA levels, and histogram of EPA/AA ratio were shown in Supplementary Fig.2. The levels of EPA and AA that predicted high EPA/AA ratio (≥ 0.41) were 54.2 µg/mL (AUC: 0.934, p＜0.01; sensitivity: 93.1%; specificity: 81.5%) and 175.7 µg/mL (AUC: 0.613, p=0.05; sensitivity: 86.2 %; specificity: 36.9 %), respectively. As shown in Table 1, high EPA/AA group was significantly older (76.1 vs. 66.1 years, P＜0.01). There were no significant differences in baseline characteristics such as sex, body mass index, hypertension, diabetes mellitus, dyslipidemia, smoking, medications, eGFR level, NT-proBNP level, and the prevalence of pre-aspiration and pre-dilation. Similarly, the prevalence of STEMI, culprit vessels, multivessel disease, and baseline TIMI grade did not differ between the groups, although the peak CK level was significantly lower in the high EPA/AA group (556 vs. 1651 U/L, P=0.03).

Fig.1.

Enrollment of the study population

Supplementary Fig.2. A) Distribution of EPA and AA levels B) Histogram of EPA/AA ratio

A) The levels of EPA and AA that predicted high EPA/AA ratio (≥ 0.41) were 54.2 µg/mL (AUC: 0.934, p＜0.01; sensitivity: 93.1%; specificity: 81.5%) and 175.7 µg/mL (AUC: 0.613, p=0.05; sensitivity: 86.2 %; specificity: 36.9 %), respectively.

B) The median EPA/AA ratio of the whole patients in this study was 0.28 (inter-quartile range 0.19-0.43). This is an analysis of cases of ACS caused by plaque rupture, and EPA/AA was shifted lower compared to the study population in the Hisayama study.

EPA, eicosapentaenoic acid; AA, arachidonic acid; ACS, acute coronary syndrome.

Table 1.Baseline characteristics

	High EPA/AA (N= 29)	Low EPA/AA (N= 66)	P-value
Age, years	76.1±9.9	66.1±12.5	＜0.01
Sex, male, %	22 (75.9%)	52 (78.8%)	0.75
Body mass index	23.2±3.6	24.1±3.1	0.21
Coronary risk factors
Hypertension, %	14 (48.3%)	31 (47.0%)	0.91
Diabetes mellitus, %	10 (34.5%)	24 (36.4%)	0.86
Dyslipidemia, %	14 (48.3%)	32 (48.5%)	0.99
Smoking, %	17 (58.6%)	50 (75.8%)	0.10
Medication
Aspirin, %	3 (10.4%)	5 (7.6%)	0.66
P2Y12 receptor inhibitor, %	1 (3.5%)	1 (1.5%)	0.56
ACEI or ARB, %	4 (13.8%)	15 (22.7%)	0.30
Calcium channel blocker, %	8 (27.6%)	11 (16.7%)	0.23
β blocker, %	2 (6.9%)	1 (1.5%)	0.17
Clinical presentation			0.13
STEMI	18 (62.1%)	51 (77.3%)
NSTE-ACS	11 (37.9%)	15 (22.7%)
Culprit vessel			0.73
LMT	0 (0%)	0 (0%)
LAD	16 (55.2%)	35 (53.0%)
LCX	2 (6.9%)	8 (12.1%)
RCA	11 (37.9%)	23 (34.9%)
Multi-vessel disease, %	17 (58.6%)	40 (60.6%)	0.86
Baseline TIMI grade			0.47
0	18 (62.1%)	38 (57.6%)
1	1 (3.5%)	6 (9.1%)
2	6 (20.7%)	8 (12.1%)
3	4 (13.8%)	14 (21.2%)
Peak CK, U/L	556 (313-2196)	1651 (538-3429)	0.03
eGFR, mL/min/1.73 m2	69.2 (50.8-78.4)	73.0 (55.0-86.3)	0.16
NT-proBNP, pg/mL	485 (122-1338)	249 (126-939)	0.23
Pre-aspiration, %	17 (58.6%)	48 (72.7%)	0.18
Pre-balloon dilation, %	7 (24.1%)	20 (30.3%)	0.54

Data are expressed as the mean±standard deviation, median (25% and 75% quartiles), or number (%).

EPA, eicosapentaenoic acid; AA, arachidonic acid; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; STEMI, ST-segment elevation myocardial infarction; NSTE-ACS, non-ST-segment elevation acute coronary syndrome; LMT, left main trunk; LAD, left anterior descending coronary artery; LCX, left circumflex artery; RCA, right coronary artery; TIMI, thrombosis in myocardial infarction; CK, creatine kinase; eGFR, estimated glomerular filtration rate; NT-proBNP, N-terminal pro-brain natriuretic peptide.

Lipid Profiles

Table 2 shows that there were no significant differences in various lipid profiles between the groups. In the high EPA/AA group, the levels of EPA were significantly greater (79.7 vs. 35.9 µg/mL, P＜0.01), while AA levels tended to be lower (148.1 vs. 163.6 µg/mL, P=0.08).

Table 2.Laboratory characteristics

	High EPA/AA (N= 29)	Low EPA/AA (N= 66)	P-value
TG, mg/dL	131.0 (88.0-219.0)	121.5 (78.8-188.5)	0.74
LDL-C, mg/dL	119.0 (108.0-152.0)	126.5 (109.0-156.3)	0.33
HDL-C, mg/dL	47.0 (39.5-55.5)	42.5 (35.8-50.0)	0.15
Non-HDL-C, mg/dL	163.0 (131.5-174.0)	151.5 (133.5-194.3)	0.69
Sd-LDL-C, mg/dL	47.6 (23.6-59.5)	38.4 (26.4-57.7)	0.62
TRL-C, mg/dL	26.0 (19.5-42.0)	22.0 (16.8-33.0)	0.10
ApoB, mg/dL	110.0 (84.5-120.0)	100.0 (86.8-124.8)	0.95
ApoC3, mg/dL	10.6 (7.2-13.9)	8.9 (6.9-11.4)	0.13
MDA-LDL, U/L	97.0 (77.5-128.0)	102.0 (75.0-119.0)	0.90
EPA/AA ratio	0.49 (0.43-0.71)	0.23 (0.15-0.29)	＜0.01
EPA, μg/mL	79.7 (57.6-109.9)	35.9 (23.0-50.3)	＜0.01
AA, μg/mL	148.1 (118.8-169.4)	163.6 (124.5-192.3)	0.08
Lipoprotein (a), mg/dL	11.3 (5.6-19.1)	11.2 (4.7-18.6)	0.69
HbA1c, %	6.0 (5.7-6.8)	6.0 (5.7-7.0)	0.94
Hs-CRP, mg/L	2.39 (0.92-3.82)	2.12 (0.94-6.09)	0.64

Data are expressed as the mean±standard deviation or median (25% and 75% quartiles).

TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; Sd-LDL-C, small dense LDL-C; TRL- C, TG-rich lipoprotein cholesterol; ApoB, Apolipoprotein B; ApoC3, apolipoprotein C3; MDA-LDL, malondialdehyde-modified LDL; EPA, eicosapentaenoic acid; AA, arachidonic acid; HbA1c, glycated hemoglobin; Hs-CRP, high-sensitivity C-reactive protein.

Oct Findings

The OCT results are listed in Table 3. The inter- and intra-observer kappa coefficients for the layered plaques were 0.740 and 0.818, respectively. The mean lesion length for all patients in this study was 25.6 mm, with an average analysis of approximately 130 cross sections done per patient. The prevalence of layered plaque was significantly higher in the high EPA/AA group than the low EPA/AA group (75.9 vs. 39.4%, P＜0.01). The prevalence of thin-cap fibroatheromas, cholesterol crystals, macrophages, grades, and microchannels were similar in the two groups, although calcified plaque was significantly higher in the high EPA/AA group (79.1 vs. 50.0 %, P＜0.01). The minimum lumen area tended to be smaller in the high EPA/AA group. There were no significant differences in the reference lumen area, percentage of stenosis, mean lipid arc, lipid length, and index between the two groups, whereas the mean calcification arc, calcification length, and calcification index were greater in the high EPA/AA group.

Table 3.Optical coherence tomography findings

	High EPA/AA (N= 29)	Low EPA/AA (N= 66)	P-value
Thin-cap fibroatheroma, %	20 (69.0%)	51 (77.3%)	0.40
Cholesterol crystal, %	20 (69.0%)	41 (62.1%)	0.36
Layered plaque, %	22 (75.9%)	26 (39.4%)	＜0.01＊
Calcified plaque, %	23 (79.3%)	33 (50.0%)	＜0.01＊
Macrophage accumulation, %	29 (100%)	65 (98.5%)	0.39
Macrophage grades, %			0.15
Grade I	1 (3.5%)	0 (0.0%)
Grade II	8 (27.6%)	15 (22.7%)
Grade III	8 (27.6%)	31 (47.0%)
Grade IV	12 (41.4%)	20 (30.3%)
Microchannel, %	15 (51.7%)	26 (39.4%)	0.27
Minimum lumen area, mm2	0.90 (0.73-1.18)	1.03 (0.83-1.45)	0.07
Mean reference lumen area, mm2	7.55 (5.61-9.23)	8.19 (5.92-9.77)	0.29
Percentage of area stenosis, %	86.4 (80.6-89.8)	84.8 (77.7-90.4)	0.54
Lesion length, mm	26.2 (22.3-30.7)	25.1 (19.0-30.9)	0.64
Mean lipid arc, °	230.3 (178.2-275.0)	199.0 (154.5-253.8)	0.15
Lipid length, mm	21.5 (15.7-26.5)	16.9 (11.9-25.7)	0.13
Lipid index	5265 (3323-6507)	3207 (2008-6317)	0.11
Mean calcification arc, °	66.5 (22.5-98.9)	0.0 (0.0-60.1)	＜0.01＊
Calcification length, mm	3.8 (0.5-6.8)	0.0 (0.0-2.4)	＜0.01＊
Calcification index	260.3 (10.5-618.7)	0.0 (0.0-124.6)	＜0.01＊

Data are expressed as number (%) or median (25% and 75% quartiles).

EPA, eicosapentaenoic acid; AA, arachidonic acid. (^＊) p＜0.05, adjusted for age.

In this study, patients were divided into three groups (Supplementary Table 1) according to the median overall EPA levels (48.9, µg/mL) and the median EPA levels in the high EPA/AA ratio group (79.7 µg/mL). The highest prevalence of layered plaques was observed in the group with EPA levels above 79.7 µg/mL, followed by the 48.9-79.6 and ＜48.9 µg/mL groups. In contrast, the prevalence of thin-cap fibroatheromas was lowest in the group with EPA levels above 79.7 µg/mL, followed by the 48.9-79.6 and ＜48.9 µg/mL groups. Fig.2 shows a representative STEMI case of plaque rupture with and without a layered plaque.

Supplementary Table 1.Comparison of age and optical coherence tomography findings at different EPA levels

	EPA levels (μg/mL)			P-value
	≥ 79.7 (N = 16)	48.9-79.6 (N = 31)	＜48.9 (N = 48)
Age, years	75.8±9.6	72.0±13.7	65.2±11.6	＜0.01
Thin-cap fibroatheroma, %	8 (50.0%)	23 (74.2%)	40 (83.3%)	0.04
Cholesterol crystal, %	11 (68.8%)	19 (61.3%)	31 (64.6%)	0.94
Layered plaque, %	12 (75.0%)	17 (54.8%)	19 (39.6%)	0.04
Calcified plaque, %	12 (75.0%)	19 (61.3%)	25 (52.1%)	0.25
Macrophage accumulation, %	16 (100%)	30 (96.8%)	48 (100%)	0.23
Macrophage grades, %				0.68
Grade I	0 (0%)	1 (3.2%)	0 (0%)
Grade II	4 (25.0%)	6 (19.4%)	13 (27.1%)
Grade III	5 (31.3%)	13 (41.9%)	21 (43.8%)
Grade IV	7 (43.8%)	11 (35.5%)	14 (29.2%)
Microchannel, %	8 (50.0%)	15 (48.4%)	18 (37.5%)	0.53

Data are expressed as the mean±standard deviation, number (%), or median (25% and 75% quartiles). EPA, eicosapentaenoic acid.

Fig.2. Cases of plaque rupture with/without layered plaque

A. OCT images of the plaque rupture from distal to proximal views (A1–A3). Case of plaque rupture with layered plaques. A1 shows a layered plaque at the culprit lesion (white arrow). A2 shows calcified plaque (^*) and plaque rupture (yellow arrow). Plaque rupture was diagnosed based on rupture of the fibrous covering and formation of a cavity inside the plaque. EPA, AA, and EPA/AA ratio were 62.2 µg/mL, 107.8 µg/mL, and 0.58, respectively.

B. Case of plaque rupture without a layered plaque. A layered plaque was not detected on the cross-sectional image from distal to proximal views of the lesion on OCT (B1-B3). B2 and B3 show plaque rupture (yellow arrow) with red thrombus. EPA, AA, and the EPA/AA ratio were 50.1 µg/mL, 140.7 µg/mL, and 0.36 respectively.

EPA, eicosapentaenoic acid; AA, arachidonic acid

Univariate and multivariate regression analyses are shown in Table 4. Multivariate logistic regression model analysis after adjusting for the EPA/AA group and age showed that high EPA/AA ratio was an independent factor for the development of layered and calcified plaques. Peak CK levels were not associated with the EPA/AA ratio or layered plaques.

Table 4.Univariate analysis and multivariate regression analyses as factors of layered, calcified plaque, or peak creatine kinase

	Univariate analysis			Multivariate analysis
	OR	95% CI	P value	OR	95% CI	P value
Layered plaque
High EPA/AA	4.84	1.81-12.93	＜0.01	5.98	2.04-17.51	＜0.01
Age	0.99	0.96-1.03	0.79	1.02	0.98-1.06	0.29
Calcified plaque
High EPA/AA	3.83	1.38-10.63	＜0.01	3.15	1.08-9.23	0.04
Age	1.03	1.00-1.07	0.04	0.98	0.94-1.02	0.26
	t		P value	t		P value
Peak CK
High EPA/AA	-2.14		0.03	-0.89		0.38
Age	-3.03		＜0.01	-2.40		0.02
Layered plaque	-0.91		0.37	-0.49		0.63
Calcified plaque	-0.83		0.41	0.00		0.99

EPA, eicosapentaenoic acid; AA, arachidonic acid; CK, creatine kinase; OR, odds ratio; CI, confidence interval.

Discussion

The main finding of this study is that the prevalence of layered and calcified plaques was significantly higher in the high EPA/AA ratio group. To the best of our knowledge, this is the first study to show an association between EPA/AA ratio and plaque characteristics in patients with plaque rupture using OCT.

Plaque healing may help patients with atherosclerosis avoid ACS, but it can lead to chronic coronary syndrome⁴³⁾. The detection of OCT-defined layered plaques is highly sensitive and specific for the in vivo identification of layered plaque patterns by histopathology²⁹⁾. In a cohort study of 105 patients with CAD, Vergallo et al. demonstrated that the prevalence of layered plaques was lower in patients with a history of recurrent ACS than in those with a history of long-term clinical stability, and that layered plaques represent a marker of long-term clinical stability⁴⁴⁾. A previous study investigating the culprit lesions in patients with ACS using OCT reported that layered plaques were more prevalent and more frequently observed in NSTE-ACS than STEMI (35.5% vs. 25.3%)⁴⁵⁾. Araki et al. showed that patients with a layered culprit plaque presented more frequently with stable angina pectoris than ACS, in a study including 313 patients with CAD (53.4% vs. 30.7%, P＜0.01)⁴⁶⁾. In our findings, the high EPA/AA group tended to have a smaller minimum lumen area and a longer lipid length, although the differences were not significant. This might support previous reports that lesions with layered plaques have smaller minimum lumen area and longer lesion length⁴⁶⁾. Pathological reports suggest that extensive calcification (sheet calcification) is a marker of plaque stability due to the low necrotic area^{47, 48)}. Histological calcification was observed more frequently in healed plaque ruptures, supporting our results ⁴⁷⁾. Dai et al. investigated 325 patients with ACS using OCT and reported that the patients with layered plaques in non-culprit lesions had more calcified plaques (52.7% vs. 34.5%, P＜0.01)⁴⁹⁾. These results are in agreement with our findings. Therefore, it has been suggested that a high EPA/AA ratio may affect healed plaque development to prevent occlusive thrombus formation.

An experimental study with administration of high dose EPA demonstrated that free EPA incorporated into the thin-cap plaque colocalized with anti-inflammatory M2 macrophage¹⁵⁾. Matsumoto et al. reported that the atherosclerotic plaques of mice administered with EPA (mean blood EPA concentration; 3542.5 µg/ml) showed a stable morphology associated with a reduced accumulation of macrophages accompanied by an increase in smooth muscle cells and collagen content¹⁴⁾. Larson et al. showed that supplementation with EPA inhibits the platelet membrane to form the coagulation cascade complex at the sites of vascular injury, thereby resulting in decreased rates of prothrombin and thrombin formation¹³⁾. Several studies have confirmed that EPA induce relaxation in isolated arteries contraction in a concentration-dependent manner^{11, 12)}. To the best of our knowledge, no studies have clearly shown an association between blood levels of EPA including whole plasma, plasma phospholipid, plasma cholesterol esters and/or erythrocyte membrane phospholipid, and biological effects on vascular cells. In addition, it remains controversial which level of EPA is important for vasoprotective effects, among the absolute amount, the percentage of total fatty acids, or the ratio to AA^{9, 50)}. While, recent evidence suggests that specialized pro-resolving mediators derived from EPA have direct cardioprotective actions in vivo⁵¹⁾. The higher EPA group in our data was significantly older, which might reflect the cumulative effect of EPA concentration on the development of layered and calcified plaques, since EPA may exert its functions after incorporation into cell membranes.

In our study, the high EPA/AA ratio group had a higher prevalence of calcification and calcification indices. In contrast to our results, several experimental studies have demonstrated that EPA suppresses arterial calcification in vitro via various mechanisms^{52, 53)}. However, the effect of EPA on calcification has not been well understood in clinical studies. Miyoshi et al. reported that a 12-month treatment with 1.8 g/day of EPA did not attenuate the progression of coronary artery calcification score which was evaluated by computed tomography in statin-treated patients (N=157) who were asymptomatic for CAD⁵⁴⁾. A population-based cross-sectional study of 1,086 men showed that serum EPA levels had a non-significant inverse association with coronary artery calcification score⁵⁵⁾. The EVAPORATE study examined changes in coronary plaques, including low-attenuation, fibrous, calcified, and non-calcified plaques in patients treated with statins plus a higher dose of EPA (4 g/day) over an 18-month period. The study showed progression of calcified plaques: EPA, 1% decrease vs. placebo, 15% increase, p=0.053 ⁵⁶⁾. In previous studies evaluating by computed tomography, coronary artery calcification scores were assessed in the whole coronary artery, whereas our study only assessed within the culprit lesion; therefore, it would be difficult to compare calcification in these studies. In addition, a period of 12-18 months may not be sufficient to assess the effect of EPA on calcification. Previous pathological studies have reported the presence of calcifications of varying sizes in more than three-quarters of plaque rupture⁴⁷⁾. In our data, the prevalence of calcified plaques was 62%, which might underestimate micro-calcified plaques at OCT resolution. The efficacy of EPA for plaque treatment in patients with plaque rupture has not been completely clarified, and further studies are needed.

Limitations

First, this was a single-center study with a relatively small sample size. Future studies should evaluate these issues in a larger number of patients. Second, the formation of vulnerable plaques that cause plaque rupture takes many years. Therefore, the analysis of lipid markers and plaques at the onset of ACS may not be sufficient to elucidate the full impact of plaques on EPA. Third, the patients in the high EPA/AA group were significantly older in this study. It is possible that age-related changes in plaques cannot be ruled out by multivariate analysis alone. The clinical significance of layered plaques in patients with ACS should be examined. Fourth, the thrombectomy procedure might modify the culprit lesion morphology, and the residual thrombus might affect the analysis of plaque characteristics. The resolution and penetration depth of OCT imaging are inadequate for accurate detection of plaque ruptures. Fifth, some iatrogenic luminal injuries caused by intracoronary thrombectomy may have been incorrectly identified as plaque ruptures. Sixth, it is difficult to distinguish thin-cap fibroatheromas from ruptured plaques with a thin-fibrous cap by OCT because thin-cap fibroatheromas can be present at a distance from the ruptured plaque or can be present continuously. Seventh, we did not measure EPA levels as a percentage of total fatty acids. Finally, only patients who underwent OCT-guided percutaneous coronary intervention were included; therefore, a selection bias may have occurred. Our findings may not be applicable to patients with renal dysfunction, severe heart failure, or ostial lesions who are not good candidates for OCT examination.

Conclusion

This study established that the prevalence of layered and calcified plaques was greater in the high EPA/AA group among patients with plaque rupture. A high EPA/AA ratio might be associated with the development of layered and calcified plaques, which may affect thrombus volume in patients with plaque rupture.

Acknowledgements

We are grateful for the valuable help of the nursing staff of the catheterization laboratory and all cardiologists at the Department of Cardiology of the Showa University Hospital for this study. We also gratefully acknowledge Motoko Ohta, Yasuki Ito, Maki Namatame, and Prof. Eisuke Inoue for technical assistance and statistical analysis. We would like to thank Editage (www.editage.com) for the English language editing.

Conflict of Interest

Shinji Koba received research funding from Denka Co. Ltd. Toshiro Shinke received research funding from Abbott Medical. The other authors have no conflicts of interest to declare.

Financial Support

The funding agencies had no role in the preparation of the manuscript.

Author Contributions

TS and SK (Shinji Koba) contributed to study design, preparation of the manuscript, data analysis, data interpretation, and critical revision of the manuscript. TA, NM, YY, SS, HT (Hideaki Tanaka), and RM contributed to data acquisition and analysis. YO, KO, KA, KN, KS, HT (Hiroaki Tsujita), SK (Seita Kondo), and ST contributed to the data acquisition. HM contributed to the data analysis and interpretation. MHY, TM, HS, and TS (Toshiro Shinke) contributed to the data interpretation, critical revision, and editing of the manuscript.

1.Supplemental Methods

1.1. Study Subjects

ACS comprised ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation ACS (NSTE-ACS). STEMI was defined as the presence of anginal symptoms (＞20 min) associated with an elevation of the ST-segment on electrocardiographic of at least 0.1 mV in two or more limb leads or at least 0.2 mV in two or more precordial leads and a rise in cardiac-specific troponin I levels. NSTE-ACS included non-STEMI (NSTEMI) and unstable angina pectoris. NSTEMI was defined as the presence of ischemic symptoms in the absence of elevation of the ST-segment on electrocardiography with elevated cardiac-specific troponin I levels. Unstable angina pectoris was defined as newly developed and accelerated chest symptoms in exertion or rest angina without a significant increase in cardiac-specific troponin I levels. The thrombosis in myocardial infarction (TIMI) flow grade of the culprit coronary artery was visually estimated during the initial CAG (TIMI0-3)¹⁾. Multivessel disease is defined as luminal stenosis of at least 70% in at least two major coronary arteries or in one coronary artery in addition to a 50% or greater stenosis of the left main trunk.

The diagnosis of hypertension was based on a history of hypertension or systolic blood pressure of ＞140 mmHg or diastolic blood pressure of ＞90 mmHg²⁾. Diabetes mellitus was diagnosed if a patient’s fasting blood glucose level was ≥ 126 mg/dL, 2-h glucose level during an oral glucose tolerance test was ≥ 200 mg/dL, random serum glucose level was ≥ 200 mg/dL, HbA1c value was ≥ 6.5%, and/or if the patient received treatment with any hypoglycemic agents²⁾. Dyslipidemia was defined as the current use of lipid-lowering medications and/or meeting the criteria of the Japan Atherosclerosis Society for fasting serum lipid levels (LDL cholesterol [LDL-c] ≥ 140 mg/dL, high-density lipoprotein cholesterol [HDL-c] ＜40 mg/dl, or triglyceride [TG] ≥ 150 mg/dL)²⁾. The body mass index was calculated as weight (kg) divided by height (m) squared. Patients who smoked at least one cigarette daily on admission were classified as current smokers.

1.2. Measurement of Biomarkers

Total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and glycated hemoglobin (HbA1c) levels were assayed using standard laboratory procedures. Serum levels of apolipoprotein (apo) were determined using an immunoturbidometric assay (Daiichi Chemical Co., Ltd. Tokyo, Japan). Serum malondialdehyde-modified LDL (MDA-LDL) levels were measured using an enzyme-linked immunosorbent assay system. The kits used for LDL-C and sd-LDL-C tests were provided by Denka Co., Ltd. (Tokyo, Japan). Serum LDL-C levels were determined using a direct homogenous assay with detergents (LDL-EX; Denka Seiken). Serum samples were frozen at −80℃ until use in the direct homogeneous assay to measure sd-LDL-C levels, as previously described³⁾. Non-HDL-c levels were estimated by subtracting the HDL-C concentration from the total cholesterol concentration. TRL-c levels were estimated by subtracting LDL-c levels from non-HDL-c⁴⁾. Lipoprotein(a) levels were measured using immunoturbidimetric assay. The high sensitivity C-reactive protein (hs-CRP) was assayed by the Dade Behring BN assay⁵⁾. The serum N-terminal pro-brain natriuretic peptide (NT-proBNP) level was measured using an Elecsys proBNP immunoassay (Roche Diagnostics, Risch, Switzerland). A serum creatinine-based formula was used to calculate the estimated glomerular filtration rate (eGFR): eGFR=194×creatinine^−1.094×age^−0.287 (×0.739 for females)⁶⁾. Peak creatine kinase (CK) was evaluated every 6 hours after the onset of symptoms for the first 2 days. A serum creatinine-based formula was used to calculate the estimated glomerular filtration rate (eGFR): eGFR=194×creatinine^−1.094×age^−0.287 (×0.739 for females)⁶⁾.

References

1)Group TS: The Thrombolysis in Myocardial Infarction (TIMI) trial. Phase I findings. N Engl J Med, 1985; 312: 932-936

2)Kinoshita M, Yokote K, Arai H, Iida M, Ishigaki Y, Ishibashi S, Umemoto S, Egusa G, Ohmura H, Okamura T, Kihara S, Koba S, Saito I, Shoji T, Daida H, Tsukamoto K, Deguchi J, Dohi S, Dobashi K, Hamaguchi H, Hara M, Hiro T, Biro S, Fujioka Y, Maruyama C, Miyamoto Y, Murakami Y, Yokode M, Yoshida H, Rakugi H, Wakatsuki A, Yamashita S, Committee for E and Clinical Management of A: Japan Atherosclerosis Society (JAS) Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017. J Atheroscler Thromb, 2018; 25: 846-984

3)Ito Y, Fujimura M, Ohta M and Hirano T: Development of a homogeneous assay for measurement of small dense LDL cholesterol. Clin Chem, 2011; 57: 57-65

4)Elshazly MB, Mani P, Nissen S, Brennan DM, Clark D, Martin S, Jones SR, Quispe R, Donnellan E, Nicholls SJ and Puri R: Remnant cholesterol, coronary atheroma progression and clinical events in statin-treated patients with coronary artery disease. Eur J Prev Cardiol, 2020; 27: 1091-1100

5)Roberts WL, Moulton L, Law TC, Farrow G, Cooper-Anderson M, Savory J and Rifai N: Evaluation of nine automated high-sensitivity C-reactive protein methods: implications for clinical and epidemiological applications. Part 2. Clin Chem, 2001; 47: 418-425

6)Imai E, Horio M, Watanabe T, Iseki K, Yamagata K, Hara S, Ura N, Kiyohara Y, Moriyama T, Ando Y, Fujimoto S, Konta T, Yokoyama H, Makino H, Hishida A and Matsuo S: Prevalence of chronic kidney disease in the Japanese general population. Clin Exp Nephrol, 2009; 13: 621-630

References

• 1)  Adkins Y and Kelley DS: Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids. J Nutr Biochem, 2010; 21: 781-792
• 2)  Mason RP, Libby P and Bhatt DL: Emerging Mechanisms of Cardiovascular Protection for the Omega-3 Fatty Acid Eicosapentaenoic Acid. Arterioscler Thromb Vasc Biol, 2020; 40: 1135-1147
• 3)  Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K and Japan EPAlisI: Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet, 2007; 369: 1090-1098
• 4)  Weinberg RL, Brook RD, Rubenfire M and Eagle KA: Cardiovascular Impact of Nutritional Supplementation With Omega-3 Fatty Acids: JACC Focus Seminar. J Am Coll Cardiol, 2021; 77: 593-608
• 5)  Kromhout D, Yasuda S, Geleijnse JM and Shimokawa H: Fish oil and omega-3 fatty acids in cardiovascular disease: do they really work? Eur Heart J, 2012; 33: 436-443
• 6)  Kromhout D, Bosschieter EB and de Lezenne Coulander C: The inverse relation between fish consumption and 20-year mortality from coronary heart disease. N Engl J Med, 1985; 312: 1205-1209
• 7)  Hu FB, Bronner L, Willett WC, Stampfer MJ, Rexrode KM, Albert CM, Hunter D and Manson JE: Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA, 2002; 287: 1815-1821
• 8)  Hu FB, Cho E, Rexrode KM, Albert CM and Manson JE: Fish and long-chain omega-3 fatty acid intake and risk of coronary heart disease and total mortality in diabetic women. Circulation, 2003; 107: 1852-1857
• 9)  Superko HR, Superko SM, Nasir K, Agatston A and Garrett BC: Omega-3 fatty acid blood levels: clinical significance and controversy. Circulation, 2013; 128: 2154-2161
• 10)  Watanabe Y and Tatsuno I: Omega-3 polyunsaturated fatty acids for cardiovascular diseases: present, past and future. Expert Rev Clin Pharmacol, 2017; 10: 865-873
• 11)  Limbu R, Cottrell GS and McNeish AJ: Characterisation of the vasodilation effects of DHA and EPA, n-3 PUFAs (fish oils), in rat aorta and mesenteric resistance arteries. PLoS One, 2018; 13: e0192484
• 12)  Omura M, Kobayashi S, Mizukami Y, Mogami K, Todoroki-Ikeda N, Miyake T and Matsuzaki M: Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation. FEBS Lett, 2001; 487: 361-366
• 13)  Larson MK, Tormoen GW, Weaver LJ, Luepke KJ, Patel IA, Hjelmen CE, Ensz NM, McComas LS and McCarty OJ: Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation. Am J Physiol Cell Physiol, 2013; 304: C273-279
• 14)  Matsumoto M, Sata M, Fukuda D, Tanaka K, Soma M, Hirata Y and Nagai R: Orally administered eicosapentaenoic acid reduces and stabilizes atherosclerotic lesions in ApoE-deficient mice. Atherosclerosis, 2008; 197: 524-533
• 15)  Sato T, Horikawa M, Takei S, Yamazaki F, Ito TK, Kondo T, Sakurai T, Kahyo T, Ikegami K, Sato S, Sato R, Jinno Y, Kawano H, Naoe S, Arita M, Kashiwagi Y and Setou M: Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques. Arterioscler Thromb Vasc Biol, 2019; 39: 1802-1816
• 16)  Mozaffarian D and Wu JH: Omega-3 fatty acids and cardiovascular disease: effects on risk factors, molecular pathways, and clinical events. J Am Coll Cardiol, 2011; 58: 2047-2067
• 17)  Back M: Omega-3 fatty acids in atherosclerosis and coronary artery disease. Future Sci OA, 2017; 3: FSO236
• 18)  Serhan CN: Pro-resolving lipid mediators are leads for resolution physiology. Nature, 2014; 510: 92-101
• 19)  Tearney GJ, Regar E, Akasaka T, Adriaenssens T, Barlis P, Bezerra HG, Bouma B, Bruining N, Cho JM, Chowdhary S, Costa MA, de Silva R, Dijkstra J, Di Mario C, Dudek D, Falk E, Feldman MD, Fitzgerald P, Garcia-Garcia HM, Gonzalo N, Granada JF, Guagliumi G, Holm NR, Honda Y, Ikeno F, Kawasaki M, Kochman J, Koltowski L, Kubo T, Kume T, Kyono H, Lam CC, Lamouche G, Lee DP, Leon MB, Maehara A, Manfrini O, Mintz GS, Mizuno K, Morel MA, Nadkarni S, Okura H, Otake H, Pietrasik A, Prati F, Raber L, Radu MD, Rieber J, Riga M, Rollins A, Rosenberg M, Sirbu V, Serruys PW, Shimada K, Shinke T, Shite J, Siegel E, Sonoda S, Suter M, Takarada S, Tanaka A, Terashima M, Thim T, Uemura S, Ughi GJ, van Beusekom HM, van der Steen AF, van Es GA, van Soest G, Virmani R, Waxman S, Weissman NJ, Weisz G and International Working Group for Intravascular Optical Coherence T: Consensus standards for acquisition, measurement, and reporting of intravascular optical coherence tomography studies: a report from the International Working Group for Intravascular Optical Coherence Tomography Standardization and Validation. J Am Coll Cardiol, 2012; 59: 1058-1072
• 20)  Wakabayashi Y, Funayama H, Ugata Y, Taniguchi Y, Hoshino H, Ako J and Momomura S: Low eicosapentaenoic acid to arachidonic acid ratio is associated with thin-cap fibroatheroma determined by optical coherence tomography. J Cardiol, 2015; 66: 482-488
• 21)  Hasegawa T, Otsuka K, Iguchi T, Matsumoto K, Ehara S, Nakata S, Nishimura S, Kataoka T, Shimada K and Yoshiyama M: Serum n-3 to n-6 polyunsaturated fatty acids ratio correlates with coronary plaque vulnerability: an optical coherence tomography study. Heart Vessels, 2014; 29: 596-602
• 22)  Kita Y, Watanabe M, Kamon D, Ueda T, Soeda T, Okayama S, Ishigami K, Kawata H, Horii M, Inoue F, Doi N, Okura H, Uemura S and Saito Y: Effects of Fatty Acid Therapy in Addition to Strong Statin on Coronary Plaques in Acute Coronary Syndrome: An Optical Coherence Tomography Study. J Am Heart Assoc, 2020; 9: e015593
• 23)  Yamano T, Kubo T, Shiono Y, Shimamura K, Orii M, Tanimoto T, Matsuo Y, Ino Y, Kitabata H, Yamaguchi T, Hirata K, Tanaka A, Imanishi T and Akasaka T: Impact of eicosapentaenoic acid treatment on the fibrous cap thickness in patients with coronary atherosclerotic plaque: an optical coherence tomography study. J Atheroscler Thromb, 2015; 22: 52-61
• 24)  Nishio R, Shinke T, Otake H, Nakagawa M, Nagoshi R, Inoue T, Kozuki A, Hariki H, Osue T, Taniguchi Y, Iwasaki M, Hiranuma N, Konishi A, Kinutani H, Shite J and Hirata K: Stabilizing effect of combined eicosapentaenoic acid and statin therapy on coronary thin-cap fibroatheroma. Atherosclerosis, 2014; 234: 114-119
• 25)  Virmani R, Kolodgie FD, Burke AP, Farb A and Schwartz SM: Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol, 2000; 20: 1262-1275
• 26)  Virmani R, Burke AP, Farb A and Kolodgie FD: Pathology of the vulnerable plaque. J Am Coll Cardiol, 2006; 47: C13-18
• 27)  Mann J and Davies MJ: Mechanisms of progression in native coronary artery disease: role of healed plaque disruption. Heart, 1999; 82: 265-268
• 28)  Arbab-Zadeh A and Fuster V: The myth of the “vulnerable plaque”: transitioning from a focus on individual lesions to atherosclerotic disease burden for coronary artery disease risk assessment. J Am Coll Cardiol, 2015; 65: 846-855
• 29)  Shimokado A, Matsuo Y, Kubo T, Nishiguchi T, Taruya A, Teraguchi I, Shiono Y, Orii M, Tanimoto T, Yamano T, Ino Y, Hozumi T, Tanaka A, Muragaki Y and Akasaka T: In vivo optical coherence tomography imaging and histopathology of healed coronary plaques. Atherosclerosis, 2018; 275: 35-42
• 30)  Arai K, Koba S, Yokota Y, Tsunoda F, Tsujita H, Kondo S, Tsukamoto S, Shoji M and Shinke T: Relationships of Fatty Acids, Delta-5 Desaturase Activity, and Lipid Profiles in Men with Acute Coronary Syndrome. J Atheroscler Thromb, 2020; 27: 1216-1229
• 31)  Ninomiya T, Nagata M, Hata J, Hirakawa Y, Ozawa M, Yoshida D, Ohara T, Kishimoto H, Mukai N, Fukuhara M, Kitazono T and Kiyohara Y: Association between ratio of serum eicosapentaenoic acid to arachidonic acid and risk of cardiovascular disease: the Hisayama Study. Atherosclerosis, 2013; 231: 261-267
• 32)  Dai J, Fang C, Zhang S, Hou J, Xing L, Li L, Wang Y, Wang J, Wang Y, Tu Y, Zhang X, Liu H, Xu M, Ren X, Ma L, Yu H, Wei G, Zhang S, Mintz GS and Yu B: Not All Plaque Erosions Are Equal: Novel Insights From 1,660 Patients With STEMI: A Clinical, Angiographic, and Intravascular OCT Study. JACC Cardiovasc Imaging, 2020; 13: 516-518
• 33)  Shin ES, Ann SH, Singh GB, Lim KH, Yoon HJ, Hur SH, Her AY, Koo BK and Akasaka T: OCT-Defined Morphological Characteristics of Coronary Artery Spasm Sites in Vasospastic Angina. JACC Cardiovasc Imaging, 2015; 8: 1059-1067
• 34)  Shin ES, Her AY, Ann SH, Balbir Singh G, Cho H, Jung EC, Shim EB, Koo BK and Akasaka T: Thrombus and Plaque Erosion Characterized by Optical Coherence Tomography in Patients With Vasospastic Angina. Rev Esp Cardiol (Engl Ed), 2017; 70: 459-466
• 35)  Nagasawa Y, Shinke T, Toh R, Ishida T, Otake H, Takaya T, Sugiyama D, Toba T, Kuroda M, Takahashi H, Terashita D, Tahara N, Shinkura Y, Uzu K, Kashiwagi D, Kuroda K, Nagano Y, Yamamoto H, Yanaka K, Tsukiyama Y and Hirata KI: The impact of serum trans fatty acids concentration on plaque vulnerability in patients with coronary artery disease: Assessment via optical coherence tomography. Atherosclerosis, 2017; 265: 312-317
• 36)  Gonzalo N, Escaned J, Alfonso F, Nolte C, Rodriguez V, Jimenez-Quevedo P, Banuelos C, Fernandez-Ortiz A, Garcia E, Hernandez-Antolin R and Macaya C: Morphometric assessment of coronary stenosis relevance with optical coherence tomography: a comparison with fractional flow reserve and intravascular ultrasound. J Am Coll Cardiol, 2012; 59: 1080-1089
• 37)  Kume T, Okura H, Yamada R, Kawamoto T, Watanabe N, Neishi Y, Sadahira Y, Akasaka T and Yoshida K: Frequency and spatial distribution of thin-cap fibroatheroma assessed by 3-vessel intravascular ultrasound and optical coherence tomography: an ex vivo validation and an initial in vivo feasibility study. Circ J, 2009; 73: 1086-1091
• 38)  Fracassi F, Crea F, Sugiyama T, Yamamoto E, Uemura S, Vergallo R, Porto I, Lee H, Fujimoto J, Fuster V and Jang IK: Healed Culprit Plaques in Patients With Acute Coronary Syndromes. J Am Coll Cardiol, 2019; 73: 2253-2263
• 39)  Janoudi A, Shamoun FE, Kalavakunta JK and Abela GS: Cholesterol crystal induced arterial inflammation and destabilization of atherosclerotic plaque. Eur Heart J, 2016; 37: 1959-1967
• 40)  Yabushita H, Bouma BE, Houser SL, Aretz HT, Jang IK, Schlendorf KH, Kauffman CR, Shishkov M, Kang DH, Halpern EF and Tearney GJ: Characterization of human atherosclerosis by optical coherence tomography. Circulation, 2002; 106: 1640-1645
• 41)  Uemura S, Ishigami K, Soeda T, Okayama S, Sung JH, Nakagawa H, Somekawa S, Takeda Y, Kawata H, Horii M and Saito Y: Thin-cap fibroatheroma and microchannel findings in optical coherence tomography correlate with subsequent progression of coronary atheromatous plaques. Eur Heart J, 2012; 33: 78-85
• 42)  Kato K, Yonetsu T, Kim SJ, Xing L, Lee H, McNulty I, Yeh RW, Sakhuja R, Zhang S, Uemura S, Yu B, Mizuno K and Jang IK: Comparison of nonculprit coronary plaque characteristics between patients with and without diabetes: a 3-vessel optical coherence tomography study. JACC Cardiovasc Interv, 2012; 5: 1150-1158
• 43)  Vergallo R and Crea F: Atherosclerotic Plaque Healing. N Engl J Med, 2020; 383: 846-857
• 44)  Vergallo R, Porto I, D’Amario D, Annibali G, Galli M, Benenati S, Bendandi F, Migliaro S, Fracassi F, Aurigemma C, Leone AM, Buffon A, Burzotta F, Trani C, Niccoli G, Liuzzo G, Prati F, Fuster V, Jang IK and Crea F: Coronary Atherosclerotic Phenotype and Plaque Healing in Patients With Recurrent Acute Coronary Syndromes Compared With Patients With Long-term Clinical Stability: An In Vivo Optical Coherence Tomography Study. JAMA Cardiol, 2019; 4: 321-329
• 45)  Russo M, Kim HO, Kurihara O, Araki M, Shinohara H, Thondapu V, Yonetsu T, Soeda T, Minami Y, Higuma T, Lee H, Fracassi F, Vergallo R, Niccoli G, Crea F, Fuster V and Jang IK: Characteristics of non-culprit plaques in acute coronary syndrome patients with layered culprit plaque. Eur Heart J Cardiovasc Imaging, 2020; 21: 1421-1430
• 46)  Araki M, Yonetsu T, Russo M, Kurihara O, Kim HO, Shinohara H, Thondapu V, Soeda T, Minami Y, Higuma T, Lee H, Kakuta T and Jang IK: Predictors for layered coronary plaques: an optical coherence tomography study. J Thromb Thrombolysis, 2020; 50: 886-894
• 47)  Mori H, Torii S, Kutyna M, Sakamoto A, Finn AV and Virmani R: Coronary Artery Calcification and its Progression: What Does it Really Mean? JACC Cardiovasc Imaging, 2018; 11: 127-142
• 48)  Jinnouchi H, Sato Y, Sakamoto A, Cornelissen A, Mori M, Kawakami R, Gadhoke NV, Kolodgie FD, Virmani R and Finn AV: Calcium deposition within coronary atherosclerotic lesion: Implications for plaque stability. Atherosclerosis, 2020; 306: 85-95
• 49)  Dai J, Fang C, Zhang S, Li L, Wang Y, Xing L, Yu H, Jiang S, Yin Y, Wang J, Wang Y, Guo J, Lei F, Liu H, Xu M, Ren X, Ma L, Wei G, Zhang S, Hou J, Mintz GS and Yu B: Frequency, Predictors, Distribution, and Morphological Characteristics of Layered Culprit and Nonculprit Plaques of Patients With Acute Myocardial Infarction: In Vivo 3-Vessel Optical Coherence Tomography Study. Circ Cardiovasc Interv, 2020; 13: e009125
• 50)  Miura K, Hughes MCB, Ungerer JPJ, Smith DD and Green AC: Absolute versus relative measures of plasma fatty acids and health outcomes: example of phospholipid omega-3 and omega-6 fatty acids and all-cause mortality in women. Eur J Nutr, 2018; 57: 713-722
• 51)  Watanabe Y and Tatsuno I: Prevention of Cardiovascular Events with Omega-3 Polyunsaturated Fatty Acids and the Mechanism Involved. J Atheroscler Thromb, 2020; 27: 183-198
• 52)  Saito Y, Nakamura K, Miura D, Yunoki K, Miyoshi T, Yoshida M, Kawakita N, Kimura T, Kondo M, Sarashina T, Akagi S, Watanabe A, Nishii N, Morita H and Ito H: Suppression of Wnt Signaling and Osteogenic Changes in Vascular Smooth Muscle Cells by Eicosapentaenoic Acid. Nutrients, 2017; 9:
• 53)  Kageyama A, Matsui H, Ohta M, Sambuichi K, Kawano H, Notsu T, Imada K, Yokoyama T and Kurabayashi M: Palmitic acid induces osteoblastic differentiation in vascular smooth muscle cells through ACSL3 and NF-kappaB, novel targets of eicosapentaenoic acid. PLoS One, 2013; 8: e68197
• 54)  Miyoshi T, Kohno K, Asonuma H, Sakuragi S, Nakahama M, Kawai Y, Uesugi T, Oka T, Munemasa M, Takahashi N, Mukohara N, Habara S, Koyama Y, Nakamura K, Ito H and Investigators P: Effect of Intensive and Standard Pitavastatin Treatment With or Without Eicosapentaenoic Acid on Progression of Coronary Artery Calcification Over 12 Months- Prospective Multicenter Study. Circ J, 2018; 82: 532-540
• 55)  Sekikawa A, Mahajan H, Kadowaki S, Hisamatsu T, Miyagawa N, Fujiyoshi A, Kadota A, Maegawa H, Murata K, Miura K, Edmundowicz D, Ueshima H and Group SR: Association of blood levels of marine omega-3 fatty acids with coronary calcification and calcium density in Japanese men. Eur J Clin Nutr, 2019; 73: 783-792
• 56)  Budoff MJ, Bhatt DL, Kinninger A, Lakshmanan S, Muhlestein JB, Le VT, May HT, Shaikh K, Shekar C, Roy SK, Tayek J and Nelson JR: Effect of icosapent ethyl on progression of coronary atherosclerosis in patients with elevated triglycerides on statin therapy: final results of the EVAPORATE trial. Eur Heart J, 2020; 41: 3925-3932