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Nikiforov et al. Proinflammatory monocyte polarization in T2DM and CHD

acute cardiovascular mortality and elevations in agreement with the Declaration of Helsinki. Written
[3]
circulating cholesterol and blood pressure . It is informed consent was obtained from all patients.
likely that this phenomenon arises from excessive
formation of reactive oxygen species in mitochondria In order to evaluate the ability of monocytes to be
due to oxidation of glucose under hyperglycemic activated, cells were isolated from whole blood using
blood conditions [4,5] . We have previously observed magnetic CD14-positive separation with MACS CD14-
a 10-25-fold increase in the oxidative modification positive microbeads (MiltenyiBiotec) and MACS
of lipoproteins in patients with Т2DM, compared to separation columns (MiltenyiBiotec). Monocytes were
[6]
healthy subjects . Macrophages capturing modified seeded into sterile 24-well culture plates at a density
6
low density lipoproteins (LDL) through scavenger- of 10 cells per well with X-Vivo serum-free medium
receptors accumulate lipids and become lipid-rich foam (Lonza) and cultured at 37 °C in CO 2 -incubator (95%
[7]
cells, thereby leading to atherogenesis . air and 5% CO 2 ).
Signs of local and systemic non-specific inflammatory Once cultured, monocytes were stimulated with
processes in atherosclerosis trace to the earliest stages 100 ng/mL of interferon-γ. Secretion of TNF-α was
[8]
of vascular lesion development . Increased oxidation of considered a marker of the pro-inflammatory response.
LDL contributes to their diffusion into the subendothelial The concentration of TNF-α in the culture medium was
space and activation of nuclear factor kappa-B (NF-kB) determined by enzyme-linked immunosorbent assay
through interaction with toll-like receptors. This process (ELISA) on day 1 after cell isolation.
[9]
leads to production of inflammatatory mediators .
However, activation of NF-kB also activates genetic Statistical processing was performed using the
programs necessary for resolution of inflammation [10] . SPSS package (SPSS Inc., USA). T-test was used to
Recent studies showed the relationship between the compare between groups.
progression of atherosclerotic plaque formation and the
ratio of pro-inflammatory (M1) and anti-inflammatory RESULTS
[11]
(M2) activated macrophages . The role of polarization
of macrophages in Т2DM is still unclear. To address As a model for investigation of changes in the immune
this, we studied the basal and induced secretion of system of studied patients, we used primary culture
the pro-inflammatory cytokine, tumor necrosis factor of monocytes isolated from the blood and evaluated
alpha (TNF-α), by monocytes isolated from the blood of cellular responses to pro-inflammatory stimuli. We
patients with newly diagnosed T2DM, compared to those revealed possible bias of monocyte polarization
of patients with coronary heart disease. towards the M1 phenotype. This procedure was
applied to non-treated T2DH patients (n = 28), CHD
METHODS patients (n = 27) and healthy subjects (n = 50) (their
clinical characteristics are given in Table 1). Cells were
stimulated with interferon-γ (100 ng/mL). Secretion of
Primary cultures of human monocytes were obtained TNF-α was measured by ELISA.
from peripheral blood of 28 non-treated patients with
newly diagnosed diabetes 2 (11 men, 17 women) and Monocytes from subjects with CHD were characterized
27 patients (20 men, 7 women) with coronary heart by a low degree of basal secretion of TNF-α [Table 2].
disease (CHD) without disturbances in carbohydrate Moreover, monocytes lacked the ability to be activated
metabolism. As a control group, 50 healthy volunteers in response to both pro- and anti-inflammatory stimuli.
without disturbances in carbohydrate and lipid metabolism On the contrary, monocytes from subjects with T2DM
were examined (25 men, 25 women) [Table 1]. exhibited a significant 3-fold increase in the basal
secretion of TNF-α as well as a 3-4-fold increase in the
The levels of glucose in the blood serum were production of TNF-α in response to stimuli.
determined by hexokinase (Thermo Scientific). The
levels of glycated hemoglobin in red blood cells were In this study, we attempted to explore the relationship
determined by the instrument immune inhibition Bekman between glycated hemoglobin (HbA1c) levels in patients
Coulter AU 680. with T2DM and susceptibility of their monocytes to
activation. The obtained data indicated that there was
None of the patients had any clinical symptoms of an obvious trend towards a direct association between
systemic inflammation. The study was approved by HbA1c levels and the monocyte basal secretion of
the local ethics committee and was carried out in TNF-α [Figure 1].

Vessel Plus ¦ Volume 1 ¦ December 28, 2017 193