DOI 10.

1007/s10517-017-3860-5
608 Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  163,  No.  5,  September,  2017  GENERAL PATHOLOGY AND PATHOPHYSIOLOGY

Adipokine and Cytokine Profiles of Epicardial

and Subcutaneous Adipose Tissue in Patients
with Coronary Heart Disease
O. V. Gruzdeva1, O. E. Akbasheva2, Yu. A. Dyleva1, L. V. Antonova1,
V. G. Matveeva1, E. G. Uchasova1, E. V. Fanaskova1, V. N. Karetnikova1,
S. V. Ivanov1, and O. L. Barbarash1
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 163, No. 5, pp. 560-563, May, 2017
Original article submitted August 23, 2016

The content of adipokines, pro- and anti-inflammatory cytokines were studied in adipocytes
isolated from epicardial and subcutaneous adipose tissue of 24 coronary heart disease pa-
tients. The content of leptin and soluble leptin receptor in adipocytes of epicardial adipose
tissue was higher by 28.6 and 56.9% and the level of adiponectin was lower by 33% than in
adipocytes of the subcutaneous fat. In culture of epicardial adipocytes, the levels of proinflam-
matory cytokines TNF-α and IL-1 were higher. Subcutaneous adipose tissue adipocytes were
characterized by higher levels of anti-inflammatory cytokines IL-10 and FGF-β. In epicardial
adipocytes of coronary heart disease patients, the concentrations of leptin, TNF-α, and IL-1
were higher, while the levels of defense regulatory molecules (adiponectin, IL-10, and FGF-β)
were lower than in subcutaneous adipocytes.
Key Words: epicardial adipose tissue; subcutaneous fat; adipocytes; cytokines

The key mechanism triggering the development of cor-
onary heart disease (CHD) is lipid metabolism distur-
bances, high content of atherogenic lipid-transporting
cholesterol forms, increased metabolic activity of the
adipose tissue producing adipokines and cytokines,
initiating, in turn, the formation of atherosclerotic
plaques and thrombotic reactions in coronary arteries
[2,3]. High concentrations of leptin and proinflamma-
tory cytokines and low concentrations of adiponectin
in the blood of patients with myocardial infarction
were previously reported [5]. Adipose tissue can be
the source of adipokines and cytokines, as virtually
all patients present with second-third degree obesity
[1]. Imbalance in the leptin—adiponectin system is
associated with unfavorable prognosis, cardiovascular
1
Research Institute of Complex Problems of Cardiovascular Diseases,
Kemerovo; 2Siberian State Medical University, Ministry of Health of
the Russian Federation, Tomsk, Russia. Address for correspon-
dence: o_gruzdeva@mail.ru. O. V. Gruzdeva
complications, and in some cases, with manifestation
of type 2 diabetes mellitus [1,5].
An important contributor to the development of
cardiovascular disease is visceral adipose tissue, partic-
ularly of epicardial location near the atria and coronary
arteries [8]. Epicardial adipose tissue (EAT) adipocytes
secrete adipokines and proinflammatory cytokines di-
rectly to the coronary arteries, thus provoking fulminant
development of atherosclerosis [13,14]. The presence
of inflammatory mediators in tissues adjacent to epi-
cardial coronary arteries can lead to activation of vas-
cular inflammation, induction of fibrotic changes in the
myocardium, instability of atheromatous plaques, and
manifestation of acute coronary events [13]. However,
the adipokine and cytokine profiles of visceral fat, spe-
cifically, of epicardial location, in CHD are less studied
than those of subcutaneous adipose tissue (SAT).
We studied the levels of adipokines, pro- and anti-
inflammatory cytokines in adipocytes isolated from
EAT and SAT of CHD patients.

0007­-4888/17/16350608 © 2017 Springer Science+Business Media New York

O. V. Gruzdeva, O. E. Akbasheva, et al. 609

MATERIALS AND METHODS proportion in complete nutrient medium M199 (Gibco)

The study included 24 CHD patients (10 men and
14 women; mean age 63.50 (57.5, 68.0) years). The
predominant cardiovascular risk factors were arterial
hyper­tension (58.3%), tobacco smoking (16.7%), dysli­
pi­demia (16.7%), type 2 diabetes mellitus (33.3%),
and excessive body weight (82.5%). The median body
weight index was 28.57 (26.47, 30.28) kg/m2. All pa-
tients signed informed consent to participation in the
study.
Adipocytes isolated from human SAT and EAT
were cultured. The cells were derived from biopsy
specimens of subcutaneous and epicardial fat (3-5 g)
collected during coronary bypass surgery. Specimens
of EAT were collected from the right heart (right
atrium and right ventricle) — zones of its greatest
presentation. Specimens of SAT were collected from
subcutaneous fat of the lower corner of the mediasti-
nal wound. Adipose tissue specimens were plunged
in Hank’s balanced saline (Sigma-Aldrich) with peni-
cillin (100 U/liter), streptomycin (100 mg/ml), and
gentamicin (50 mg/ml) and transported to laboratory.
Isolation of adipocytes from adipose tissue was carried
out under sterile conditions in a laminar box, class II
protection (BOV-001-AMC (aseptic medical systems,
Mias Medical Equipment Plant) as described previ-
ously [6]. Fragments of EAT and SAT (1-2 mm3) were
incubated for 30 min at 37oC in collagenase I solu-
tion (0.5 mg/ml; Invitrogen) with 200 nM adenosine
(Sigma-Aldrich). After incubation, collagenase was in-
hibited by adding 10% fetal calf serum (Gibco) in 1:1
with 1% HEPES buffer, 1% L-glutamine solution with
penicillin and streptomycin, 0.4% amphotericin B (all
components from Gibco), and glucose in the final con-
centration of 5 mmol/liter.
Floating fragments of enzyme-processed adipose
tissue were transferred onto a sterile filter with 100 µ
pores (Falcon), washed (stirring carefully) in M199
complete culture medium, and the volume of the tube
contents with the cells was brought to 5 ml, after
which the material was centrifuged (2 min, 200g). The
resultant adipocytes were placed into a new tube and
the volume was brought to 1 ml with culture medium,
after which adipocytes were counted in Goryaev’s
chamber. Cell viability was evaluated as described
previously [11].
Adipocytes (20×105) were put into a well of a
sterile 24-well plate (Greiner) and the volume of the
well was put to 1 ml with culture medium. The cells
were cultured for 48 h, the medium was replaced
with a fresh portion after 24 h. The medium was
carefully collected from the bottom of the well before
the beginning and every 24 h of culturing for sub-
sequent measurements of cytokines and adipokines
by ELISA.
The levels of adipokines (adiponectin, leptin, and
leptin receptor) and cytokines (TNF-α, IL-1, IL-10,
and FGF-β) in EAT and SAT were measured by ELISA
using BioVendor and eBioscience test systems.
The data were processed by Mann—Whitney
and Wilcoxon nonparametric tests. Statistical hypoth-
eses were verified at the critical level of significance

TABLE 1. Content of Adiponectin, Leptin, and Soluble Leptin Receptor in Adipocyte Cultures from EAT and SAT (Me (Q1;Q3)

EAT adipocytes SAT adipocytes

Parameter day 1 day 2 day 1 day 2 p

1 2 3 4

Leptin, ng/ml 0.56 0.44 0.40 0.24 p1,2=0.002

(0.52;0.59) (0.39;0.50) (0.28;0.40) (0.19;0.25) p3,4=0.007
p1,3=0.0001
p2,4=0.001
Soluble leptin
receptor, ng/ml 4.62 4.61 1.99 1.97 p1,2=0.3
(2.12;6.04) (2.40;5.91) (1.43;4.8) (1.62;3.90) p1,3=0.003
p2,4=0.0002
Adiponectin, mg/ml 0.15 0.20 0.20 0.25 p1,2=0.027
(0.11;0.17) (0.13;0.20) (0.13;0.23) (0.21;0.38) p3,4=0.022
p1,3=0.009
p2,4=0.0003
610 Bulletin  of  Experimental  Biology  and  Medicine,  Vol.  163,  No.  5,  September,  2017  GENERAL PATHOLOGY AND PATHOPHYSIOLOGY

p<0.05. The results were presented as the median with
the upper and lower quartiles (Me (Q1;Q3).
RESULTS
Visceral and subcutaneous adipocytes differed by ex-
pression of adipocyte and cytokine genes [12]. Secre-
tion of EAT and SAT adipocyte factors into culture
medium was studied. The content of leptin and soluble
leptin receptor were higher in EAT adipocytes. On day
1 of EAT adipocyte culturing, the content of leptin and
its receptors was 28.6 and 56.9% higher, respectively
(Table 1). On day 2 of culturing, the differences were
more pronounced: 45.5 and 57.3% for leptin and leptin
receptors, respectively. The increase of leptin con-
centration could cause negative shifts in adipocytes
and cardiomyocytes, specifically, disorders in cellular
energy homeostasis with accumulation of free fatty
acid oxidation products with cytotoxic effects in the
cells [7].
The leptin concentration decreased during cultur-
ing in the EAT adipocyte culture medium (by 21.4%)
and in subcutaneous fat (by 40%), which could be due
to poor viability of adipocytes in vitro (Table 1).
In contrast to leptin, adiponectin concentration on
day 1 was 33.3% lower in epicardial adipocytes than
in subcutaneous cells. Adiponectin content increased
during culturing: by 33.3% in epicardial adipocytes
and by 25% in SAT adipocytes. On day 2 of culturing,
the level of adiponectin was still lower in epicardial
adipocyte culture medium. Adiponectin was characte­
rized by anti-inflammatory and antiatherogenic effect.
TABLE 2. Content of TNF-α, IL-10, IL-1, and FGF-β in Adipocyte Cultures from EAT and SAT (Me (Q1;Q3)
The decrease of its concentration in EAT was associ-
ated with coronary artery atherosclerosis [10]. Ac-
cording to our data, 90% patients had atherosclerotic
involvement of two or three coronary arteries.
The levels of proinflammatory cytokines TNF-α
and IL-1 were higher in epicardial adipocyte culture
(Table 2). Activation of the proinflammatory potential
in the ischemic myocardium zone could induce fibrous
changes in cardiomyocytes, leading to their pathologi-
cal remodeling [4].
Subcutaneous adipocytes were characterized by
higher content of protective molecules — anti-inflam-
matory cytokine IL-10 and reparation and angiogen-
esis stimulator FGF-β. By contrast, the content of IL-
10 in epicardial adipocyte culture was 60% lower on
day 1 of culturing and 50.3% lower on day 2. The
content of FGF-β in epicardial adipocyte culture was
51% lower on day 1. By day 2 of culturing the con-
centration of FGF-β dropped by 81.7% in epicardial
adipocytes and by 83% in subcutaneous adipocytes.
However, on day 2 of culturing the content of FGF-β
in subcutaneous adipocytes was 55.8% higher than in
epicardial adipocytes.
Hence, EAT adipocytes in CHD differ by adipo-
kine and cytokine profile from SAT adipocytes. Epicar-
dial adipocytes of CHD patients are characterized by
marked imbalance of the adipokine status with higher
leptin concentration and lower adiponectin level, with
predominating proinflammatory cytokines and deficit
of anti-inflammatory IL-10. Subcutaneous adipocytes
of CHD patients release a grater amount of defense
regulatory molecules (adiponectin, anti-inflammatory

EAT adipocytes SAT adipocytes

Parameter day 1 day 2 day 1 day 2 p

1 2 1 2

TNF-α, pg/ml 780.8 761.5 673.3 700.5 p3,4=0.0005

(560.1;833.15) (715.9;795.6) (503.85;736.9) (650.3;747.5) p1,3=0.001
p2,4=0.002
IL-1, pg/ml 11.83 11.90 10.95 11.22 p1,3=0.003
(11.29;12.89) (11.32;12.06) (10.84;12.33) (10.32;11.24) p2,4=0.0003
IL-10, pg/ml 5.86 6.70 14.75 13.69 p1,3=0.019
(5.36;12.55) (5.54;17.89) (6.12;18.20) (7.61;25.01) p2,4=0.002
FGF-β, pg/ml 198.50 33.66 405.80 76.14 p1,2=0.0001
(142.9;261.7) (23.70;60.71) (319.7;548.0) (49.36;87.99) p3,4=0.0001
p1,3=0.001
p2,4=0.001
O. V. Gruzdeva, O. E. Akbasheva, et al.
cytokine IL-10, reparation and angiogenesis stimulator
FGF-β) than epicardial adipocytes. Adiponectin and
FGF-β are synergic. According to some data, adipo-
nectin is an essential mediator of metabolic vascular
reactions mediated by FGF. Decrease of adiponectin
content in epicardial adipocytes is a prognostically
unfavorable sign indicating discontinuation of FGF-β
cardioprotective effect. Study of the unique materi-
al — visceral EAT, located in the immediate vicin-
ity of coronary arteries — has detected pathogenetic
significance of changes in the adipokine and cytokine
status of adipocytes in CHD.
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