International Journal of Cardiology xxx (xxxx) xxx

Contents lists available at ScienceDirect

International Journal of Cardiology

journal homepage: www.elsevier.com/locate/ijcard

Cholesterol crystals in non-culprit plaques of STEMI patients: A 3-vessel

OCT study
Zhifeng Qin a, b, 1, Muhua Cao a, b, 1, Xiangwen Xi a, b, Yanwen Zhang a, b, Zhuozhong Wang a, b,
Suhong Zhao a, b, Yanan Tian a, b, Qinglu Xu a, b, Huai Yu a, b, Jinwei Tian a, b, *, Bo Yu a, b, *
a
Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
b
The Key Laboratory of Myocardial Ischemia, Ministry of Education, Harbin, China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Cholesterol crystals (CCs) are regular microstructures found within the necrotic core of athero­
Cholesterol crystal sclerotic plaques and have been hypothesized to be related to plaque destabilization. We attempted to investigate
Non-culprit plaque the potential association between CCs and non-culprit plaque vulnerability in patients with ST-segment elevated
ST-segment elevation myocardial infarction
myocardial infarction (STEMI) and study morphological features of CCs in ruptured non-culprit plaques.
Optical coherence tomography
Methods: A total of 261 patients with ST-segment elevation myocardial infarction who underwent 3-vessel optical
coherence tomography (OCT) imaging were included. Non-culprit plaques were divided into two groups ac­
cording to the presence or absence of CCs in the plaque to compare the morphological characteristics of the
plaques. The differences in parameters of the non-culprit plaque CCs were explored between ruptured plaques
and unruptured plaques.
Results: Totally, 530 non-culprit plaques (29 ruptured plaques and 501 unruptured plaques) were identified by
OCT. The incidence of CCs was 21.1%. Compared with non-culprit plaques without CCs, those with CCs had a
larger lipid burden. Macrophages (p < 0.001) and spotty calcification (p = 0.002) were more frequently observed
in non-culprit plaques with CCs. The frequency of CCs was significantly higher (p = 0.001) and the CCs were
larger (p = 0.046) and more superficial (p = 0.005) in ruptured non-culprit plaques than in unruptured non-
culprit plaques. The maximum lipid arc and fibrous cap thickness were independent predictors of plaque
rupture, but the presence of CCs was not.
Conclusions: Non-culprit plaques with CCs have more vulnerable features. CCs are more frequently found in
ruptured non-culprit plaques and larger and more superficial CCs are associated with plaque rupture.

1. Introduction myocardial infarction (STEMI) patients have multivessel coronary ar­

Cholesterol crystals (CCs) are an important kind of microstructure in
atherosclerotic plaques and are believed to be involved in plaque
destabilization [1]. The presence of CCs in culprit plaques, which is
often reported in the research community, has been found to be asso­
ciated with a higher prevalence of vulnerable plaque characteristics [2].
Several optical coherence tomography (OCT) studies have reported that
patients with culprit or non-culprit plaque CCs have worse outcomes
than those without culprit plaque CCs [2]. Usui et al. found that the
presence of an OCT-detected low- intensity area without attenuation
with CC within a non-culprit plaque is associated with future major
adverse cardiac events [3]. More than half of ST-segment elevation
tery disease, and the evaluation of non-culprit plaques cannot be ignored
[4,5]. However, the effect of CCs in non-culprit plaques on plaque sta­
bility remains unclear. The morphologic characteristics of non-culprit
CCs in ruptured plaques have not been fully demonstrated. CCs are
observed as thin, linear regions of high intensity, and sharp-bordered
structures on OCT [6]. Whether their volume, cross-sectional area,
depth, width and other specific parameters affect the stability of the
plaque has not been elucidated. Thus, the aim of this study was to assess
(1) non-culprit plaque features according to the presence or absence of
CCs and (2) specific morphological differences of CCs in ruptured versus
unruptured plaques.

* Corresponding authors at: Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, 150086 Harbin, China.
E-mail addresses: tianjinweidr2009@163.com (J. Tian), dryu_hmu@163.com (B. Yu).
1
Zhifeng Qin and Muhua Cao contributed equally to this paper.

https://doi.org/10.1016/j.ijcard.2022.06.016
Received 19 April 2022; Received in revised form 6 June 2022; Accepted 10 June 2022
Available online 12 June 2022
0167-5273/© 2022 Elsevier B.V. All rights reserved.

Please cite this article as: Zhifeng Qin, International Journal of Cardiology, https://doi.org/10.1016/j.ijcard.2022.06.016
Z. Qin et al.
2. Method
2.1. Study population
This was a retrospective, single-center cohort study analyzing pa­
tients with STEMI who underwent successful OCT imaging of 3 major
epicardial coronary arteries at the 2nd Affiliated Hospital of Harbin
Medical University. Between January 2018 and February 2019, 343
consecutive eligible patients were screened. Eighty-two of these patients
were excluded for the following reasons: poor image quality (n = 13), in-
stent thrombosis or neoatherosclerosis (n = 16), balloon dilatation
before performing OCT and thromboembolism (n = 19), and OCT seg­
ments that did not include all non-culprit plaques (n = 34). Finally, 261
patients with STEMI were enrolled in this study. Preintervention OCT of
the culprit vessel was performed before treatment of the culprit lesion,
while OCT of the non-culprit arteries was performed after percutaneous
coronary intervention of the culprit lesion. STEMI was defined using the
criteria in current guidelines [7]. This study was conducted in accor­
dance with the Declaration of Helsinki and approved by the institutional
ethical committee. All patients provided written informed consent.
2.2. Coronary angiography analysis
Coronary angiography was performed via the radial or femoral
approach after intracoronary injection of heparin and nitroglycerin. The
patients were pretreated with 300 mg aspirin and 300 mg clopidogrel
before the intervention procedure. Quantitative coronary angiography
(QCA) was performed using offline software (QAngio XA 7.3, Leiden,
The Netherlands). Catheter calibration was performed before analysis of
the coronary angiography images to ensure that the results were
2
International Journal of Cardiology xxx (xxxx) xxx
accurate. The reference vessel diameter (RVD), minimal luminal diam­
eter (MLD), and diameter stenosis (DS) were calculated by an experi­
enced operator who was blinded to the patient clinical information and
OCT findings. The percentage of diameter stenosis was calculated as
follows: (Reference vessel diameter - Minimal lumen diameter)/ Refer­
ence vessel diameter) × 100%. All lesions were defined according to the
AHA/ACC guidelines [8].
2.3. OCT image acquisition and analysis
OCT imaging of all lesions was performed using a commercially
available C7-XR/ILUMIEN OCT system (Abbott Vascular, Santa Clara,
California). All the images were analyzed using offline proprietary
software (QIvus Research Edition 3.1, Leiden, The Netherlands) by 2
experienced investigators who were blinded to the patient information.
If there was a disagreement between the two investigators, the third
analyst read the images independently to finally reach a consensus.
The culprit plaques were identified using established criteria [9].
Non-culprit plaques were identified as a region with at least 30% lumen
area stenosis estimated by OCT compared with the mean reference area
and loss of the normal architecture of the vessel wall [10]. The plaque
morphologies and various intraplaque microstructures were qualita­
tively and quantitatively analyzed at 0.2-mm intervals. CCs are defined
as thin, linear regions of high intensity, sharp-bordered and sometimes
stacked structures on OCT images [6,10]. In this study, we measured
specific CC parameters. Considering that a plaque may contain multiple
CCs, we measured the length, width, thickness, depth, cross-sectional
area and volume of each CC in the same plaque. Because the length of
a CC is too short to be measured precisely, it was roughly calculated as
the number of frames in which CCs appeared multiplied by the interval
Fig. 1. Measurement methods for CCs.
Figure 1 shows how we measured the maximum
width A, depth B, area C and thickness D of CCs. The
maximum length was roughly calculated as the
number of frames in which CCs appeared multiplied
by the interval distance between two adjacent frames
(0.2 mm). The volume of a CC was roughly estimated
as the sum of the cross-sectional area of each frame of
CCs multiplied by the interval distance between two
adjacent frames (0.2 mm). CC = cholesterol crystal.
Z. Qin et al. International Journal of Cardiology xxx (xxxx) xxx

distance between two adjacent frames (0.2 mm). The depth of a CC was Table 1
defined as the minimum distance between the CC and the lumen. Baseline Clinical Characteristics.
Measurements of the width, depth, thickness and cross-sectional area of Characteristics All patients Patients with Patients P
non-culprit CCs are shown in Fig. 1. The volume of a CC was roughly (n = 261) non-culprit CCs without non- value
estimated as the sum of the cross-sectional area of each frame of CCs (n = 86) culprit CCs
(n = 175)
multiplied by the interval distance between two adjacent frames (0.2
mm). Finally, the maximum length, maximum thickness, maximum Male, n (%) 194 (74.3) 67(77.9) 127(72.6) 0.354
width, maximum cross-sectional area, maximum volume and minimum Age, years 58.4 ± 11.2 59.6 ± 10.7 57.7 ± 11.5 0.201

depth of multiple CCs within a plaque were recorded. Other definitions

of OCT plaque morphologies were derived from previous consensus Risk factors
Current smoker, 112 (42.9) 39(45.3) 73(41.7) 0.577
documents and major OCT studies (Supplementary materials) [6,10,11].
n (%)
The intraobserver and interobserver reproducibility values of CC iden­ Current drinker, 89 (34.1) 32(37.2) 57(32.6) 0.458
tification were excellent (κ, 0.93 and 0.90, respectively). Plaques had to n (%)
be at least 5 mm apart to be considered two separate plaques. Hypertension, n 101 (38.7) 37(43.0) 64(36.6) 0.314
(%)
Diabetes, n (%) 62 (23.8) 24(27.9) 38(21.7) 0.269
2.4. Statistical analysis Prior AMI, n (%) 20 (7.7) 12(14.0) 8(4.6) 0.007
Prior stroke, n 22 (8.4) 8(9.3) 14(8.0) 0.722
All statistical analyses were analyzed using SPSS version 25.0 (SPSS, (%)
IBM, Armonk, NY, USA) and R V.4.0.5 (R Foundation for Statistical
Computing, Vienna, Austria). The continuous variables were first tested Laboratory data
for normality and homogeneity of variance. The Kolmogorov–Smirnov LDL-C, mg/dL 112.0 ± 109.2 ± 37.6 113.3 ± 41.8 0.453
test was used to assess the normality of continuous data. The results 40.5
HDL-C, mg/dL 50.2 ± 12.9 46.7 ± 11.2 51.8 ± 13.4 0.004
were recorded as the mean ± standard deviation (SD) for normally
TC, mg/dL 182.9 ± 176.3 ± 44.4 185.9 ± 48.8 0.138
distributed variables and as the median (25th–75th percentiles) for 47.6
nonnormally distributed variable data. Categorical variables were TG, mg/dL 164.5 ± 161.3 ± 138.9 166.0 ± 125.5 0.788
recorded as counts and percentages. Intraobserver and interobserver 129.6
hs-CRP, mg/dL 5.0 ± 4.0 4.9 ± 3.8 5.0 ± 4.1 0.795
differences were quantified using the κ coefficient of agreement for CC
eGFR, mL/min/ 85.9 ± 26.3 83.3 ± 24.7 87.2 ± 27.1 0.255
identification. Comparisons of non-culprit plaques with or without CCs 1.732
were carried out using generalized estimating equations (GEEs) taking
into account the within-subject correlation of multiple plaques in a Values n (%), mean ± SD; AMI = acute myocardial infarction; CCs = cholesterol
crystals; eGFR = estimated glomerular filtration rate; HDL-C = high-density li­
single patient. Similarly, the same method was used to compare non-
poprotein cholesterol; hs-CRP = hypersensitive C-reactive protein; LDL-C = low-
culprit CC morphological characteristics in ruptured plaques and
density lipoprotein cholesterol; TC = total cholesterol; TG = triglyceride.
unruptured plaques, considering that a patient may have multiple non-
culprit plaques with CCs. Univariate and multivariate GEE log-binomial
regression models were used to identify factors that were independently Table 2
associated with plaque rupture. Variables exhibiting a p value <0.05 in Angiographic Findings Comparing Non-culprit Plaques with versus without CCs.
the univariate analysis were tested in the multivariate analysis. A p All non-culprit Non-culprit Non-culprit P value
value <0.05 was considered indicative of statistical significance. plaques (n = plaques without plaques with CCs (GEE)
530) CCs (n = 418) (n = 112)
3. Results Location 0.201
LAD 154(29.1) 114(27.3) 40(35.7)
3.1. Clinical characteristics RCA 191(36.0) 156(37.3) 35(31.3)
LCX 185(34.9) 148(35.4) 37(33.0)
MLD, 1.96 ± 0.63 1.99 ± 0.62 1.85 ± 0.64 0.036
A total of 261 patients with STEMI who underwent OCT of 3 main mm
vessels were enrolled in this study; 261 culprit plaques and 530 non- RVD, 3.00 ± 0.68 3.01 ± 0.70 2.94 ± 0.61 0.468
culprit plaques were identified by OCT. CCs were detected in 110 of mm
261 culprit plaques (42.1%), and the incidence of CCs in non-culprit DS, % 35.25 ± 13.30 34.47 ± 12.30 38.16 ± 16.23 0.027
plaques was 21.1% (112 of 530 non-culprit plaques). The patients Values n (%), mean ± SD, CCs = cholesterol crystals; DS = diameter stenosis;
were divided into two groups based on the presence or absence of CCs in LAD = left anterior descending artery; LCX = left circumflex artery; MLD =
the non-culprit plaques. The baseline characteristics are shown in minimal lumen diameter; RCA = right coronary artery; RVD = reference vessel
Table 1. The average age of the participants was 58.4 years (SD 11.2) diameter.
and 194 patients (74.3%) were male. There was no significant difference
in baseline characteristics between the two groups except for history of with or without CCs, while stenosis was more severe in the CC group.
myocardial infarction and level of high-density lipoprotein cholesterol The results of angiographic findings between ruptured and unruptured
(HDL–C). In addition, the baseline characteristics were well matched non-culprit plaques are shown in the supplementary material (Supple­
between the patients with and without a ruptured non-culprit lesion and mentary Table 2).
are shown in Supplementary Table 1. Patients with at least one ruptured
non-culprit plaque had a higher prevalence of diabetes (p = 0.005).
3.3. OCT findings of non-culprit plaques
3.2. QCA findings of non-culprit plaques
The OCT analyses of the non-culprit plaques are summarized in
The QCA analyses of non-culprit plaques are summarized in Table 2. Table 3. The incidence of plaque rupture was higher in the CC group
The distribution frequencies of non-culprit plaques in the left anterior than in the non-CC group (11.6% vs. 3.8%). Fibrous plaques were more
descending artery (LAD), right coronary artery (RCA), and left circum­ frequently observed in the non-CC group than in the CC group (53.6%
flex artery (LCX) were similar between the non-culprit plaques with and vs. 33.0%, p < 0.001) while the incidence of lipid rich plaques was lower
without CCs. The RVD was comparable between the non-culprit lesions (34.7% vs. 62.5%, p < 0.001). The lipid length was significantly longer

3
Z. Qin et al. International Journal of Cardiology xxx (xxxx) xxx

Table 3 (65.5% vs. 7.4%, p < 0.001), CCs (44.8% vs. 19.8%, p = 0.001),
OCT Findings Comparing Non-culprit Plaques with versus without CCs. macrophage accumulation (93.1% vs. 48.9%, p < 0.001), and thrombus
All non- Non-culprit Non-culprit P value (86.2% vs. 2.6%, p < 0.001) were more often observed in ruptured
culprit plaques plaques with (GEE) group than in the unruptured group.
plaques (n = without CCs (n CCs (n = 112) Moreover, we explored the specific morphological differences in the
530) = 418)
non-culprit plaque CCs between the ruptured and unruptured plaques
Non-culprit 29(5.5) 16(3.8) 13(11.6) 0.001 (Fig. 2). The incidence of CCs in the non-culprit plaques was higher in
rupture the ruptured group than in the other group (44.8% vs. 19.8%, p =
Fibrous plaque 261(49.2) 224(53.6) 37(33.0)
0.001). Furthermore, the maximum single CC volume (0.0232 mm3 vs.
<0.001
Fibrocalcific 54(10.2) 49(11.7) 5(4.5) 0.044
plaque 0.0081 mm3, p = 0.046) of the ruptured group seemed larger than that
Lipid-rich 215(40.6) 145(34.7) 70(62.5) <0.001 of the unruptured group. There was no significant difference in the
plaque maximum length, maximum width or maximum thickness between the
Lipid length, 10.2 ± 6.4 9.4 ± 5.6 11.7 ± 7.6 0.017
two groups. The distance from the lumen surface of CCs in the ruptured
mm
FCT, μm 89.5 ± 34.5 89.7 ± 34.0 89.1 ± 35.8 0.722 group was shorter than that in the unruptured group (191.2 ± 138.4 μm
Max lipid arc, 247.6 ± 80.0 235.7 ± 75.4 272.2 ± 84.1 0.001 vs. 321.0 ± 187.4 μm, p = 0.005).
◦

TCFA 56(10.6) 39(9.3) 17(15.2) 0.173

Minimal lumen 4.1 ± 2.2 4.3 ± 2.2 3.6 ± 2.0 0.007
3.4. Univariate and multivariate GEE log-binomial regression of plaque
area, mm2 rupture
Macrophage 272(51.3) 185(44.3) 87(77.7) <0.001
Microchannel 256(48.3) 192(45.9) 64(57.1) 0.065 In the univariate analysis, the maximum lipid arc, fibrous cap
Spotty 76(14.3) 50(12.0) 26(23.2) 0.002
thickness (FCT), macrophages and the presence of CCs were associated
calcification
Thrombus 38(7.2) 29(6.9) 9(8.0) 0.683 with plaque rupture. After adjusting for confounding factors, the
maximum lipid arc and FCT were still associated with plaque rupture.
Values n (%), mean ± SD, CCs = cholesterol crystals; FCT = fibrous cap thick­
However, the presence of CCs was not an independent predictor of a
ness; GEE = generalized estimating equation; TCFA = thin-cap fibroatheroma.
ruptured plaque. The results are shown in Table 4.

(11.7 ± 7.6 mm vs. 9.4 ± 5.6 mm, p = 0.017), and the maximum lipid
arc was larger (272.2 ± 84.1◦ vs. 235.7 ± 75.4◦ , p = 0.001) in the CC
group than in the non-CC group. Macrophages (77.7% vs. 44.3%, p <
0.001) and spotty calcifications (23.2% vs. 12.0%, p = 0.002) were more
frequently found in the non-culprit plaques with CCs. The differences in
OCT findings between the ruptured and unruptured non-culprit plaques
are shown in the supplementary material (Supplementary Table 3). The
lipid length (13.4 ± 7.6 mm vs. 9.7 ± 6.1 mm, p = 0.006) and maximum
lipid arc (298.4 ± 61.9◦ vs. 240.0 ± 79.7◦ , p < 0.001) were significantly
larger in the ruptured group than in the unruptured group. Lipid rich
plaque (96.6% vs. 37.3%, p < 0.001), thin-cap fibroatheroma (TCFA)
4. Discussion
In this study, we found that (1) Non-culprit plaques with CCs had
more vulnerable features of local inflammation and lipid burden and
that (2) CCs were more frequently found in ruptured non-culprit plaques
and larger and more superficial CCs were associated with previous
rupture.
4.1. Pathogenesis and physical triggers of CCs
The formation of CCs was associated with the disruption of the

Fig. 2. CC Morphological Characteristics of Non-culprit Plaques of Ruptured Plaques and Unruptured Plaques.
Quantitative analysis of non-culprit plaque CCs showed a higher incidence and larger maximum single CC volume in plaques with rupture than in those without
rupture. The distance from the lumen surface of CCs in the ruptured group was shorter than that in the unruptured group. CC = cholesterol crystal.

4
Z. Qin et al. International Journal of Cardiology xxx (xxxx) xxx

Table 4 response via NLRP3 inflammasome protein [19]. Intracellular crystals

Univariate and Multivariate GEE Log-Binomial Regression of Plaque Rupture.
Variable Univariate Analysis Multivariable Analysis
OR (95% CI) P value OR (95% CI)
Max lipid arc, ◦
1.010 <0.001 1.008
(1.005–1.015) (1.002–1.015)
FCT, μm 0.965 <0.001 0.969
(0.947–0.983) (0.952–0.986)
Minimal lumen 0.915 0.292
area, mm2 (0.776–1.079)
Macrophage 13.916 3.029
<0.001
(3.349–57.824) (0.387–23.713)
Microchannel 0.995 0.990
(0.441–2.245)
Spotty 0.891 0.848
calcification (0.275–2.887)
CC 3.431 0.001 1.162
(1.613–7.300) (0.454–2.978)
Values are OR (95% CI). CC = cholesterol crystal; FCT = fibrous cap thickness;
GEE = generalized estimating equation; OR = odds ratio.
equilibrium between esterified cholesterol and free cholesterol.
Cholesterol ester hydrolase enzymes convert esterified cholesterol to
free cholesterol, while acyl-coenzyme A cholesterol acyltransferase 1
converts free cholesterol back to the esterified state. Once the balance is
disrupted, free cholesterol accumulates in the extracellular matrix and
becomes supersaturated, leading to crystallization [12,13]. In addition,
dying foam cells and red blood cells are rich in free cholesterol [14].
After these cells release their contents into the extracellular space, local
physical properties change, and CCs are likely to form [13]. High-
density lipoprotein plays a critical role in the process of reverse trans­
porting cholesterol and dissolving CCs directly [15–17]. Inflammation
and CCs promote each other. A recent study showed that chronic
inflammation accelerated CC formation [18]. Likewise, CCs could
induce arterial inflammation by activating NLRP3, leading to the
secretion of IL-1b [19]. Additionally, CCs can drive inflammation by
activating Syk and PI3 kinase in specific protein pathways [20]. HDL
dysfunction caused by inflammation can result in free cholesterol
accumulation in the extracellular space and promote the formation of
CCs [21]. The baseline HDL-C analysis of patients in this study also
verified this mechanism. Physical factors also influenced cholesterol
crystallization. In addition to the saturation we just mentioned, physical
triggers of CCs include temperature, pH, and hydration status [13]. A
previous OCT study found a higher rate of plaque rupture in patients
who developed acute coronary syndrome (ACS) in winter than in those
with onset in other seasons [22]. This implied a potential link between
cholesterol crystallization and plaque rupture at low temperatures. In
vitro experiments revealed that pH is another physical factor that affects
cholesterol crystallization, with an elevated pH promoting crystalliza­
tion. Because the hydration of the cholesterol molecule could impart
certain water properties, the peak volume increased rapidly by up to
45% when cholesterol crystallized [23]. These physical factors may
trigger cholesterol crystallization alone or in combination.
4.2. Non-culprit plaque CCs, lipid deposition and plaque vulnerability
In our study, the incidence of non-culprit plaque CCs was 21.1%,
which was comparable to that in other observational studies [24,25].
Although CCs were less frequent in non-culprit plaques than in culprit
plaques, we support the hypothesis that they may weaken the stability of
the plaque, similar to culprit plaque CCs. We found that the non-culprit
plaque CC group had a higher prevalence of lipid-rich plaques than the
other group. Non-culprit plaques with CCs had a greater lipid burden,
more microstructures and more severe vessel stenosis. The formation of
atherosclerosis begins with the migration of LDL under arterial endo­
thelial cells. Macrophages phagocytose oxidized LDL into foam cells. In
foam cells, cholesterol crystallizes and initiates a local inflammatory
can lead to apoptosis of foam cells and attract more macrophages to the
site of inflammation by chemotaxis and establish a vicious cycle [26,27].
The foam cells die and release their content, and these materials
P value
intermix with cellular debris and ultimately form the lipid-rich necrotic
0.008 core, the major hallmark of a vulnerable plaque [28]. This explains why
<0.001
non-culprit plaques with CCs had more macrophages and a greater lipid
burden. The appearance of non-culprit CCs may also imply extensive
lipid deposition and plaque instability in the coronary tree. We supposed
that CCs and extensive lipid deposition may have an additive effect on
0.291
plaque stability. Plaque rupture may be favored by many factors, and the
combination of CCs and a large lipid pool or a necrotic core may be one
of the major causes. It is difficult for CCs alone to reach the rupture
threshold of non-culprit plaques in the absence of large lipid pools. We
confirmed this hypothesis by multivariate logistic regression analysis.
0.754
The presence of CCs was not an independent risk factor for plaque
rupture, but the characteristics of large lipid pools, such as the maximum
lipid arc and thin fibrous cap, were independent predictors. Regarding
calcification, although the direct interaction between CCs and calcifi­
cation is unclear, their initial formation processes are similar. One study
suggested that apoptotic bodies and necrotic debris released during the
death of macrophages contribute to the early stages of both intimal and
medial calcification [29]. Thus, spotty calcifications were more
frequently found in non-culprit plaques with CCs than in those without
CCs. The influence of CCs on plaque stability indicates that non-culprit
plaques are potentially at risk of rupture.
4.3. High-risk morphological characteristics of non-culprit plaque CCs
We further measured the morphological parameters of CCs in non-
culprit plaques and found that the CCs in ruptured plaques were
larger, more frequent and more superficial than those in the control
group. That is, maybe not all CCs are high-risk. Only specific morpho­
logical CCs contributed to plaque instability. As mentioned before,
liquid cholesterol expands quickly in volume by up to 45% when it
crystallizes [23]. CC content had been confirmed to be an independent
predictor of thrombus in an autopsy study [30]. The expansion of CCs
may cause indirect intraplaque biomechanical changes in addition to
direct mechanical effects such as cutting fibrous tissues and disrupting
the plaque cap. A virtual histology intravascular ultrasound study
demonstrates that structural stress is highly variable within plaques and
associated with plaque composition. Great differences in the material
properties of adjacent tissues generate stress concentrations, or weak
points, within the atherosclerotic plaque and vessel wall [31]. CCs are
dense crystals that differ greatly from surrounding tissue, and this
property contributes to plaque instability. Another fluid dynamics
model based on high-resolution OCT demonstrated that CCs at the pla­
que shoulder imposed the highest peak circumferential stress on the
fibrous cap and that peak circumferential stress was proportional to the
volume of CCs [32]. This change in stress has a strong potential to induce
plaque rupture. Therefore, a large volume of CCs may cause more severe
tearing of fibrous tissues [33]. A previous study of culprit lesions
confirmed that superficial CCs were a positive independent predictor for
plaque rupture [34]. We also observed this phenomenon in non-culprit
plaques. The tips of the superficial CCs can more easily penetrate the
fibrous caps. Moreover, mechanical stress increases when the intima
contains CCs. All of these factors imply that plaques with superficial CCs
are prone to rupture. Generally, this suggested that these morphological
characteristics of CCs were a high-risk phenotype and that the
morphology of non-culprit CCs may be analyzed as an indicator of risk
stratification of atherosclerotic plaques. Although these phenomena
may also occur in culprit plaques, it is difficult to observe and measure
CCs due to the rupture of culprit plaques causing content to flow out.
Thus, risk stratification according to the presence of CCs in non-culprit
plaques may be a new method worth exploring.

5
Z. Qin et al.
4.4. Limitations
Our study has the following limitations. First, this was a retrospec­
tive, observational single-center study with a limited sample size.
Moreover, there were only 29 non-culprit plaques with rupture, so we
adjusted for confounders at the plaque level. Second, OCT has a mod­
erate sensitivity for detecting CCs [34,35]. Although investigators have
gone through a considerable amount of training, it is still impossible to
find all CCs. In addition to some tiny CCs that are extremely difficult to
detect on OCT images because of the attenuation of OCT signals, the
content of plaques that have ruptured into the lumen also contains CCs.
Thus, the number of CCs may be underestimated in these cases. Finally,
we only screened STEMI patients, so the results may not be suitable for
the overall ACS population.
5. Conclusion
Characteristics of vulnerable plaques such as larger lipid burden,
macrophage accumulation and spotty calcification were seen more
frequently in non-culprit plaques with CCs in STEMI patients. CCs are
more frequently found in ruptured non-culprit plaques and larger and
more superficial CCs were associated with previous rupture.
Funding
This work was supported by grants from the National Natural Science
Foundation of China (Grant Nos. U21A20391 and 81971715 to J.W.T.),
the Fok Ying-Tong Education Foundation for Young Teachers (171032
to J.W.T.). and the HMU Marshal Initiative Funding (HMUMIF-21020 to
J.W.T.).
Disclosures
The authors have no conflicts of interest to disclose.
Author contributions
Zhifeng Qin, Muhua Cao, Bo Yu, and Jinwei Tian: study concept and
design. Zhifeng Qin, Xiangwen Xi and Yanwen Zhang: acquisition of
data. Zhifeng Qin: analysis and interpretation of data and drafting of the
manuscript. Yanan Tian, Qinglu Xu, Huai Yu and Suhong Zhao: critical
revision of the manuscript for intellectual content. Zhifeng Qin and
Zhuozhong Wang: statistical analysis. Jinwei Tian: obtaining funding.
All authors gave final approval and agreed to be accountable for all
aspects of the work, ensuring integrity and accuracy.
Acknowledgments
None.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijcard.2022.06.016.
References
[1] S. Nishimura, S. Ehara, T. Hasegawa, K. Matsumoto, J. Yoshikawa, K. Shimada,
Cholesterol crystal as a new feature of coronary vulnerable plaques: an optical
coherence tomography study, J. Cardiol. 69 (2017) 253–259.
[2] K. Fujiyoshi, Y. Minami, K. Ishida, A. Kato, A. Katsura, Y. Muramatsu, et al.,
Incidence, factors, and clinical significance of cholesterol crystals in coronary
plaque: an optical coherence tomography study, Atherosclerosis. 283 (2019)
79–84.
[3] E. Usui, M. Matsumura, G.S. Mintz, Z. Zhou, M. Hada, M. Yamaguchi, et al.,
Clinical outcomes of low-intensity area without attenuation and cholesterol
crystals in non-culprit lesions assessed by optical coherence tomography,
Atherosclerosis. 332 (2021) 41–47.
6
International Journal of Cardiology xxx (xxxx) xxx
[4] D.W. Park, R.M. Clare, P.J. Schulte, K.S. Pieper, L.K. Shaw, R.M. Califf, et al.,
Extent, location, and clinical significance of non-infarct-related coronary artery
disease among patients with ST-elevation myocardial infarction, Jama. 312 (2014)
2019–2027.
[5] B. Ibanez, S. James, S. Agewall, M.J. Antunes, C. Bucciarelli-Ducci, H. Bueno, et al.,
2017 ESC guidelines for the management of acute myocardial infarction in patients
presenting with ST-segment elevation: the task force for the management of acute
myocardial infarction in patients presenting with ST-segment elevation of the
European Society of Cardiology (ESC), Eur. Heart J. 39 (2018) 119–177.
[6] M. Araki, S.J. Park, H.L. Dauerman, S. Uemura, J.S. Kim, C. Di Mario, et al., Optical
coherence tomography in coronary atherosclerosis assessment and intervention,
Nat. Rev. Cardiol. (2022), https://doi.org/10.1038/s41569-022-00687-9.
[7] P.T. O’Gara, F.G. Kushner, D.D. Ascheim, D.E. Casey Jr., M.K. Chung, J.A. de
Lemos, et al., 2013 ACCF/AHA guideline for the management of ST-elevation
myocardial infarction: a report of the American College of Cardiology Foundation/
American Heart Association task force on practice guidelines, Circulation. 127
(2013) e362–e425.
[8] T.J. Ryan, D.P. Faxon, R.M. Gunnar, J.W. Kennedy, S.B. King 3rd, F.D. Loop, et al.,
Guidelines for percutaneous transluminal coronary angioplasty. A report of the
American College of Cardiology/American Heart Association task force on
assessment of diagnostic and therapeutic cardiovascular procedures (subcommittee
on percutaneous transluminal coronary angioplasty), Circulation. 78 (1988)
486–502.
[9] H. Jia, F. Abtahian, A.D. Aguirre, S. Lee, S. Chia, H. Lowe, et al., In vivo diagnosis
of plaque erosion and calcified nodule in patients with acute coronary syndrome by
intravascular optical coherence tomography, J. Am. Coll. Cardiol. 62 (2013)
1748–1758.
[10] G.J. Tearney, E. Regar, T. Akasaka, T. Adriaenssens, P. Barlis, H.G. Bezerra, et al.,
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. 59 (2012) 1058–1072.
[11] F. Prati, E. Regar, G.S. Mintz, E. Arbustini, C. Di Mario, I.K. Jang, et al., Expert
review document on methodology, terminology, and clinical applications of optical
coherence tomography: physical principles, methodology of image acquisition, and
clinical application for assessment of coronary arteries and atherosclerosis, Eur.
Heart J. 31 (2010) 401–415.
[12] A. Janoudi, F.E. Shamoun, J.K. Kalavakunta, G.S. Abela, Cholesterol crystal
induced arterial inflammation and destabilization of atherosclerotic plaque, Eur.
Heart J. 37 (2016) 1959–1967.
[13] A. Vedre, D.R. Pathak, M. Crimp, C. Lum, M. Koochesfahani, G.S. Abela, Physical
factors that trigger cholesterol crystallization leading to plaque rupture,
Atherosclerosis. 203 (2009) 89–96.
[14] F.D. Kolodgie, A.P. Burke, G. Nakazawa, Q. Cheng, X. Xu, R. Virmani, Free
cholesterol in atherosclerotic plaques: where does it come from? Curr. Opin.
Lipidol. 18 (2007) 500–507.
[15] J.F. Oram, R.M. Lawn, ABCA1. The gatekeeper for eliminating excess tissue
cholesterol, J. Lipid Res. 42 (2001) 1173–1179.
[16] N. Wang, D. Lan, W. Chen, F. Matsuura, A.R. Tall, ATP-binding cassette
transporters G1 and G4 mediate cellular cholesterol efflux to high-density
lipoproteins, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 9774–9779.
[17] C.W. Adams, Y.H. Abdulla, The action of human high density lipoprotein on
cholesterol crystals. Part 1. Light-microscopic observations, Atherosclerosis. 31
(1978) 465–471.
[18] Y. Baumer, N.N. Mehta, A.K. Dey, T.M. Powell-Wiley, W.A. Boisvert, Cholesterol
crystals and atherosclerosis, Eur. Heart J. 41 (2020) 2236–2239.
[19] P. Duewell, H. Kono, K.J. Rayner, C.M. Sirois, G. Vladimer, F.G. Bauernfeind, et al.,
NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol
crystals, Nature. 464 (2010) 1357–1361.
[20] E.M. Corr, C.C. Cunningham, A. Dunne, Cholesterol crystals activate Syk and PI3
kinase in human macrophages and dendritic cells, Atherosclerosis. 251 (2016)
197–205.
[21] Moya M. de la Llera, F.C. McGillicuddy, C.C. Hinkle, M. Byrne, M.R. Joshi,
V. Nguyen, et al., Inflammation modulates human HDL composition and function
in vivo, Atherosclerosis. 222 (2012) 390–394.
[22] J. Shibuya, N. Kobayashi, K. Asai, M. Tsurumi, Y. Shibata, S. Uchiyama, et al.,
Comparison of coronary culprit lesion morphology determined by optical
coherence tomography and relation to outcomes in patients diagnosed with acute
coronary syndrome during winter -vs- other seasons, Am. J. Cardiol. 124 (2019)
31–38.
[23] G.S. Abela, K. Aziz, Cholesterol crystals cause mechanical damage to biological
membranes: a proposed mechanism of plaque rupture and erosion leading to
arterial thrombosis, Clin. Cardiol. 28 (2005) 413–420.
[24] L. Xing, T. Higuma, Z. Wang, A.D. Aguirre, K. Mizuno, M. Takano, et al., Clinical
significance of lipid-rich plaque detected by optical coherence tomography: a 4-
year follow-up study, J. Am. Coll. Cardiol. 69 (2017) 2502–2513.
[25] M. Cao, L. Zhao, X. Ren, T. Wu, G. Yang, Z. Du, et al., Pancoronary plaque
characteristics in STEMI caused by culprit plaque Erosion versus rupture: 3-vessel
OCT study, J. Am. Coll. Cardiol. Img. 14 (2021) 1235–1245.
[26] Y.J. Geng, J.E. Phillips, R.P. Mason, S.W. Casscells, Cholesterol crystallization and
macrophage apoptosis: implication for atherosclerotic plaque instability and
rupture, Biochem. Pharmacol. 66 (2003) 1485–1492.
[27] G. Kellner-Weibel, W.G. Jerome, D.M. Small, G.J. Warner, J.K. Stoltenborg, M.
A. Kearney, et al., Effects of intracellular free cholesterol accumulation on
macrophage viability: a model for foam cell death, Arterioscler. Thromb. Vasc.
Biol. 18 (1998) 423–431.
Z. Qin et al.
[28] G.S. Abela, Cholesterol crystals piercing the arterial plaque and intima trigger local
and systemic inflammation, J. Clin. Lipidol. 4 (2010) 156–164.
[29] C.M. Shanahan, Inflammation ushers in calcification: a cycle of damage and
protection? Circulation. 116 (2007) 2782–2785.
[30] G.S. Abela, K. Aziz, A. Vedre, D.R. Pathak, J.D. Talbott, J. Dejong, Effect of
cholesterol crystals on plaques and intima in arteries of patients with acute
coronary and cerebrovascular syndromes, Am. J. Cardiol. 103 (2009) 959–968.
[31] V. Thondapu, C.V. Bourantas, N. Foin, I.K. Jang, P.W. Serruys, P. Barlis,
Biomechanical stress in coronary atherosclerosis: emerging insights from
computational modelling, Eur. Heart J. 38 (2017) 81–92.
[32] Y. Luo, D. Cui, X. Yu, S. Chen, X. Liu, H. Tang, et al., Modeling of mechanical stress
exerted by cholesterol crystallization on atherosclerotic plaques, PLoS One 11
(2016), e0155117.
International Journal of Cardiology xxx (xxxx) xxx
[33] Z. Teng, A.J. Brown, P.A. Calvert, R.A. Parker, D.R. Obaid, Y. Huang, et al.,
Coronary plaque structural stress is associated with plaque composition and
subtype and higher in acute coronary syndrome: the BEACON I (biomechanical
evaluation of atheromatous coronary arteries) study, Circul. Cardiovasc. Imag. 7
(2014) 461–470.
[34] Y. Katayama, A. Tanaka, A. Taruya, M. Kashiwagi, T. Nishiguchi, Y. Ozaki, et al.,
Feasibility and clinical significance of in vivo cholesterol crystal detection using
optical coherence tomography, Arterioscler. Thromb. Vasc. Biol. 40 (2020)
220–229.
[35] H. Jinnouchi, Y. Sato, S. Torii, A. Sakamoto, A. Cornelissen, R.R. Bhoite, et al.,
Detection of cholesterol crystals by optical coherence tomography, EuroInterven.:
J. EuroPCR Collab. Working Group Intervent. Cardiol. Eur. Soc. Cardiol. 16 (2020)
395–403.