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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 4, July/August 2021
AFRICA
205
The patients’ data were obtained and the average for each person
was calculated.
Ethical approval for the study was granted by the ethics
committee of ShanXi Cardiovascular Hospital. All participants
gave written informed consent and the study adhered to the
Declaration of Helsinki.
Fasting venous blood samples were collected in the ward
between 06:00 and 07:00 on the day after hospitalisation. Blood
samples were put into the centrifuge tube without anticoagulant
and centrifuged at 3 000 rpm for 15 minutes. The serum was
separated and the serum oestradiol level was determined with
chemiluminescence on a Beckman Coulter unicell DXL 800
immunoassay system.
During thoracotomy, five to 10 g EAT was taken from the
initial segment of the right atrioventricular sulcus, close to the
right coronary artery, before cardiopulmonary bypass (CPB).
The samples were stored in liquid nitrogen for future analysis.
Total RNA was extracted using an RNA extraction kit
(Qiagen, 205111) following the manufacturer’s instructions.
RT-qPCR analysis was done using the SYBR GreenER qPCR
kit (Takara, RR820A) following the manufacturer’s instructions.
Relative gene expression was determined using the 2
-ΔΔ
Ct method.
The results for each gene came from 30 independent repeated
measurements (
n
= 30/group). Primer sequences are shown in
Table 1.
To determine the presence of aromatase in EAT and compare
its levels in CHD and non-CHD patients, samples were analysed
using a human CYP19A1 ELISA kit (Cusabio Biotech, Life
Sciences Advanced Technologies). The analysis was done in
three independent replicates (
n
= 30/group).
Statistical analysis
Measurements are presented as mean
±
SD and patient
proportions as percentages. The Spearman correlation test was
used for correlation analysis. The
t
-test was used for intergroup
comparison of measured data and the chi-squared test was used
for intergroup comparison of counted data. A
p-
value
≤
0.05
indicated statistical significance. Data were analysed on SPSS
version 26.
Results
A total of 60 patients were included in this study, with a median
age of 59.17
±
11.66 years in the CHD group and 57 years
(52–66) in the control group. Hypertension was more prevalent
in the CHD group than in the control group. Low-density
lipoprotein cholesterol level and left ventricular ejection fraction
were lower in the CHD group relative to the control group (
p
≤
0.05). Other indicators did not vary significantly between the two
groups (Table 2).
To exclude the effects of serum oestrogen differences on
coronary artery lesions, oestrogen levels were measured by
chemiluminescence in the blood samples collected before CPB.
This analysis did not reveal significant differences in serum
oestrogen levels in the two groups (
p
= 0.7011, Fig. 1A).
RT-qPCR analysis indicated that relative to the non-CHD
group, EAT aromatase levels were significantly lower in the
CHD group (
p <
0.0001, Fig. 1B). ELISA showed that EAT
aromatase protein levels were also significantly lower in the
CHD group (
p <
0.0001, Fig. 1C).
Correlative analysis revealed no correlation between
aromatase mRNA and protein levels in the control versus CHD
groups (
r
control
= –0.069;
p
control
= 0.717;
r
CHD group
= –0.057;
p
CHD group
= 0.764) (Fig. 2A, B). There was a negative correlation between
aromatase protein content and SYNTAX score in the CHD
patients, hence, the higher the SYNTAX score, the lower the
aromatase protein content (correlation coefficient = –0.430,
p
=
0.018, Fig. 2C).
Discussion
Based on clinical and experimental investigation, we report for
the first time that aromatase level negatively correlated with
CHD severity. Since EAT and VAT have the same embryological
origin, EAT can be considered the visceral adipose depot in the
heart.
12
There is no fascial structure between EAT, the adjacent
myocardium and the vascular walls, and high-density adipose
tissue can directly infiltrate into the cardiomyocytes, making
contact with the adventitia of the coronary arteries.
13
This offers
an important structural basis for the endocrine role of adipose
tissue.
Coronary atherosclerotic plaques are reported to mainly
occur in arterial segments surrounded by EAT,
14
while
intramyocardial coronary artery segments are largely unaffected
by atherosclerosis.
15
EAT volume is proposed as a biomarker for
subclinical atherosclerosis, independent of other coronary artery
disease risk factors.
12
Based on these facts, we hypothesised that
EAT plays a critical role in the pathogenesis of coronary artery
disease.
Table 1.The primers of related genes for aromatase RT-qPCR
Name
Sequences (5
′→
3
′
)
Aromatase forward
TGGAAATGCTGAACCCGATAC
Aromatase reverse
AATTCCCATGCAGTAGCCAGG
Internal reference GAPDH forward CCACCCATGGCAAATTCCAATGGCA
Internal reference GAPDH reverse TCTAGACGGCAGGTCAGGTCCACC
PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehy-
drogenase
Table 2. Basic parameters of the patients
Parameters
Non-CHD group
(
n
= 30)
CHD group
(
n
= 30)
χ
2
/
t p
-value
Hypertension,
n
(%)
11 (34.4)
21 (65.6)
6.696 0.010
Hyperlipidaemia,
n
(%)
16 (47.1)
18 (52.9)
0.271 0.602
Diabetes,
n
(%)
5 (35.7)
9 (64.3)
1.491 0.222
Smoking,
n
(%)
18 (58.1)
13 (41.9)
1.669 0.196
Age, years
59.03
±
9.90
59.30
±
13.36 0.088 0.930
BMI, kg/m
2
26.39
±
2.62
27.19
±
2.71 1.163 0.250
SBP, mmHg
126.80
±
18.09 130.43
±
18.48 0.770 0.445
LDL-C, mmol/l
3.11
±
0.85
2.67
±
0.66 2.202 0.032
HDL-C, mmol/l
1.15
±
0.31
1.16
±
0.30 0.042 0.967
Scr, μmol/l
75.22
±
13.48
78.78
±
13.97 1.004 0.319
LVEF, %
64.77
±
5.81
44.10
±
9.22 10.384 < 0.001
LVEDD, mm
46.47
±
6.44
47.87
±
11.92 0.566 0.574
Values are presented as mean
±
SD or number (%).
BMI, body mass index; SBP, systolic blood pressure; LDL-C, low-density lipo-
protein cholesterol; HDL-C, high-density lipoprotein cholesterol; Scr, serum
creatinine; LVEF, left ventricular ejection fraction; LVEDD, left ventricular
end-diastolic diameter.