Hindawi Publishing Corporation

BioMed Research International

Volume 2016, Article ID 9582430, 13 pages
http://dx.doi.org/10.1155/2016/9582430

Review Article
Macrophages and Their Role in Atherosclerosis:
Pathophysiology and Transcriptome Analysis

Yuri V. Bobryshev,1,2,3 Ekaterina A. Ivanova,4 Dimitry A. Chistiakov,5

Nikita G. Nikiforov,1,6 and Alexander N. Orekhov1,6,7
1
Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow 125315, Russia
2
Faculty of Medicine, School of Medical Sciences, University of New South Wales, Kensington, Sydney, NSW 2052, Australia
3
School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia
4
Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium
5
Department of Molecular Genetic Diagnostics and Cell Biology, Institute of Pediatrics, Research Center for Children’s Health,
Moscow 119991, Russia
6
Institute for Atherosclerosis, Skolkovo Innovation Center, Moscow 143025, Russia
7
Department of Biophysics, Biological Faculty, Moscow State University, Moscow 119991, Russia

Correspondence should be addressed to Ekaterina A. Ivanova; kate.ivanov@gmail.com

Received 29 March 2016; Revised 29 May 2016; Accepted 22 June 2016

Academic Editor: Tomasz Guzik

Copyright © 2016 Yuri V. Bobryshev et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Atherosclerosis can be regarded as a chronic inflammatory state, in which macrophages play different and important roles.
Phagocytic proinflammatory cells populate growing atherosclerotic lesions, where they actively participate in cholesterol
accumulation. Moreover, macrophages promote formation of complicated and unstable plaques by maintaining proinflammatory
microenvironment. At the same time, anti-inflammatory macrophages contribute to tissue repair and remodelling and plaque
stabilization. Macrophages therefore represent attractive targets for development of antiatherosclerotic therapy, which can aim to
reduce monocyte recruitment to the lesion site, inhibit proinflammatory macrophages, or stimulate anti-inflammatory responses
and cholesterol efflux. More studies are needed, however, to create a comprehensive classification of different macrophage
phenotypes and to define their roles in the pathogenesis of atherosclerosis. In this review, we provide an overview of the current
knowledge on macrophage diversity, activation, and plasticity in atherosclerosis and describe macrophage-based cellular tests for
evaluation of potential antiatherosclerotic substances.

1. Introduction includes oxidation, enzymatic processing, desialylation, and

aggregation. These modifications render the lipoprotein par-
Atherosclerosis is a chronic inflammatory disease triggered ticles proinflammatory and induce an immune response lead-
by lipid retention in the arterial wall [1]. Certain areas of ing to the formation of circulating LDL-containing immune
arteries, such as branching points and bends, are especially complexes that are highly atherogenic [3]. Macrophages play
prone to atherosclerotic lesion development due to local a decisive role at all stages of atherosclerotic lesion progres-
disturbance of endothelial function. In such areas, circulating sion [4, 5]. It is widely accepted that circulating monocyte-
lipoprotein particles can penetrate into the arterial wall and derived cells are recruited to the atherosclerotic lesion site
accumulate in the subendothelial proteoglycan-rich layer of (Figure 1), where they differentiate into macrophages. A num-
the arterial wall intima. According to current understanding, ber of recent studies, however, challenged this paradigm by
low-density lipoprotein (LDL), especially in its modified demonstrating that most tissue macrophages develop inde-
form, serves as a primary source of lipid accumulation in pendently of monocyte input from precursor cells present in
atherosclerotic lesions [2]. Atherogenic modification of LDL adult tissues [6]. Interestingly, subendothelial intimal layer
2 BioMed Research International

At the lesion site, monocytes differentiate to macrophages

that actively take part in the immune response by engulfing
pathogens and damaged cells via phagocytosis and releasing
proinflammatory factors. On the other hand, macrophages
are also responsible for the resolution of the inflamma-
tory response and tissue remodelling. The classical system
regarded macrophages as terminally differentiated cells that
are constantly renewed by monocytes newly recruited from
(a) circulation. This understanding was based primarily on trac-
ing radiolabelled differentiating monocytes/macrophages in
mice during inflammatory response. However, more recent
studies have demonstrated that the ontogeny of tissue
macrophages is more complex, and a large proportion of
these cells derives from resident precursors [6].
Studying of monocyte/macrophage heterogeneity is chal-
lenging, because different subpopulations of these cells
defined by expression of specific markers do not completely
(b) (c) overlap in mice and humans. In both organisms, circulating
monocytes can be divided into several distinct types based on
Figure 1: Adhesion (a) and penetration (b, c) of blood monocytes the expression of surface molecules and chemokine receptors
into the intima of the human aorta. Scanning Electron Microscopy
[11]. In humans, monocytes positive for CD14 and negative for
(SEM). Scale bars = 15 𝜇m (a) and 5 𝜇m (b, c).
CD16 surface antigens are the most prevalent and are referred
to as classical monocytes [12, 13]. Like murine proinflamma-
tory (LY6Chi ) monocytes, these cells express CC-chemokine
of human arterial wall contains a population of pluripotent receptor 2 (CCR2). Monocytes positive for CD16 can be
pericyte-like cells that can differentiate into various cell further divided into 2 subsets: CD14+ CD16++ (nonclassical)
types including phagocytes, positive for macrophage marker and CD14++ CD16+ (intermediate). Although both subclasses
CD68 [7]. Macrophages in atherosclerotic lesions actively can produce proinflammatory factors, their functions in the
participate in lipoprotein ingestion and accumulation giving organism are different. Nonclassical monocytes have antiviral
rise to foam cells filled with lipid droplets. Accumulation activity and selectively produce proinflammatory factors
of foam cells contributes to lipid storage and atherosclerotic in response to viral particles and nucleic acid-containing
plaque growth. Macrophages populating the atherosclerotic complexes, patrol the tissues, and are likely to be responsible
plaque have a decreased ability to migrate, which leads to for the local immune response [14]. Intermediate monocytes
failure of inflammation resolution and to further progression are capable of producing large amounts of proinflammatory
of the lesion into complicated atherosclerotic plaque [8]. At molecules, such as tumor necrosis factor in response to
this stage, macrophages contribute to the maintenance of the stimulation [15]. Studying the distinct subpopulations of
local inflammatory response by secreting proinflammatory monocytes and their roles in the pathogenesis of atheroscle-
cytokines and chemokines and producing reactive oxygen rosis may help in developing novel therapeutic approaches
species. Dying macrophages are responsible for necrotic specifically targeting different stages of the disease progres-
core formation in progressing plaques [9]. The key role sion.
that macrophages play in the pathogenesis of atherosclerosis It has been noted that the number of circulating proin-
makes them an attractive target for therapy development. flammatory monocytes is significantly increased in animal
Several possibilities have been considered, including inhibit- models of atherosclerosis, such as Apoe−/− mice in compar-
ing monocyte/macrophage recruitment to growing lesions, ison to control animals [16]. Hypercholesterolaemia seems to
stimulating cholesterol efflux and diminishing lipid storage, promote the proliferation of haematopoietic stem and pro-
and taking advantage of macrophage plasticity and the ability genitor cells and to enhance their sensitivity to granulocyte-
to polarize towards pro- or anti-inflammatory phenotypes macrophage colony-stimulating factor (GM-CSF). On the
[5]. contrary, production of high-density lipoprotein (HDL),
which promotes cholesterol efflux and protects against
2. Mononuclear Phagocyte System and atherosclerosis, reverses this phenotype [17].
the Role of Macrophages During haematopoiesis, monocyte differentiation into
macrophages is triggered mainly by two growth factors: GM-
According to the classical view, monocytes and macrophages CSF and macrophage colony-stimulating factor (M-SCF)
form a continuous system, the mononuclear phagocyte [18]. Differentiation of circulating monocytes can be induced
system, which plays a central role in the innate immune by various stimuli, most importantly, in response to infection
response [10]. Circulating monocytes are recruited to the or aseptic inflammation. The latter process plays an impor-
sites of injury or pathogen invasion by specific signals, tant role in the pathogenesis of atherosclerosis [19]. Fatty
including cytokines and chemokines released by tissue cells. streaks represent the early stages of atherosclerotic lesion
BioMed Research International 3

development. It has been demonstrated that monocytes can M2. M1 macrophages differentiate in response to toll-like
be recruited to fatty streaks and can penetrate into the arterial receptor (TLR) and interferon-𝛾 signalling and can be
wall due to the increased endothelial permeability linked to induced by the presence of pathogen-associated molecular
the local endothelial dysfunction. In mice, both proinflam- complexes (PAMPs), lipopolysaccharides, and lipoproteins.
matory and patrolling monocytes can be recruited to growing These cells secrete proinflammatory factors, such as tumor
atherosclerotic lesions by P- and E-selectin-dependent rolling necrosis factor- (TNF-) 𝛼, interleukin-1𝛽 (IL-1𝛽), IL-12,
followed by intercellular adhesion molecule 1- (ICAM1-) and IL-23, and chemokines CXCL9, CXCL10, and CXCL11.
and vascular cell adhesion molecule 1- (VCAM1-) depen- Proinflammatory macrophages produce high levels of reac-
dent adhesion [20]. Proinflammatory monocyte migration tive oxygen species (ROS) and nitric oxide (NO) that also
into the arterial wall is mediated by CCR2, CCR5, and contribute to the development of the inflammatory response
CX3 C-chemokine receptor 1 (CX3 CR1) signalling. Corre- (Table 1) [28]. M2 macrophages that have anti-inflammatory
spondingly, inhibition of these molecules in Apoe−/− murine properties are induced in response to Th2-type cytokines IL-
model of atherosclerosis prevented monocyte recruitment 4 and IL-13 and secrete anti-inflammatory factors, such as
and atherosclerotic lesion growth [21]. Chemokines can be IL-1 receptor agonist and IL-10. Macrophages correspond-
produced by activated endothelial cells at the lesion site, as ing to M1 and M2 types were described in atherosclerotic
well as by intimal macrophages and resident arterial wall cells. lesions. Proinflammatory (M1) macrophages were enriched
Proinflammatory monocytes penetrating into the arterial in progressing plaques, and M2 macrophages were present in
wall differentiate into macrophages and contribute to the regressing plaques, where they were involved in tissue repair
inflammatory process and lesion development [16]. The role and remodelling [28].
of patrolling monocytes in the disease pathogenesis is less Recent studies have demonstrated that the bipolar M1/M2
clear. They participate in phagocytosis and might differentiate classification does not accurately describe the macrophage
into dendritic cells [22]. diversity [26]. Therefore, additional classes of macrophages
Monocytes differentiating into macrophages demonstrate were distinguished depending on activation stimuli. Some
a number of morphological and structural changes, including authors proposed to divide the M2 type into several sub-
enlargement, increase of organelles numbers, intensification groups depending on the activation stimuli and protein
of metabolism, enhanced expression of surface receptors, expression pattern. M2a macrophages induced by IL-4 and
and altered sensitivity to signalling molecules. Differentiating IL-13 express high levels of CD206, IL-1 receptor agonist
monocytes have increased lysosomal enzyme activity, which (IL1RN). M2b macrophages can be induced by TLR signalling
prepares them for active phagocytosis and digestion of the and immune complexes, as well as IL-1R ligands [27]. They
produce both anti-inflammatory (IL-10) and proinflamma-
engulfed material [23]. Importantly, macrophages that popu-
tory (IL-6, TNF-𝛼) cytokines. M2c macrophages that can
late atherosclerotic lesions have a decreased ability to migrate.
be induced by IL-10, transforming growth factor-𝛽 (TGF-𝛽),
This contributes to the failure of inflammation resolution
and glucocorticosteroids possess strong anti-inflammatory
and to the formation of complicated plaques [5, 24]. In such properties and produce pentraxin-3 (PTX3), TGF-𝛽, and
plaques, different types of immune cells, as well as resident IL-10. They express Mer receptor kinase (MERTK) and
cells of the arterial wall, participate in the inflammatory are responsible for clearance of apoptotic cells [29]. M2d
process by secreting proinflammatory factors and matrix- macrophages differentiated in response to TLR signalling
degrading proteases. The increased cell death leads to the through the adenosine A2A receptor were demonstrated to
formation of necrotic core in the progressing plaque. On have angiogenic properties that can play a role in tumor pro-
the other hand, recruitment of monocytes to the arterial gression and atherosclerotic plaque growth [30]. This classifi-
wall can also be important for inflammation resolution and cation, however, can be further broadened to include species-
atherosclerotic lesion regression [25]. specific macrophage types. For instance, Mox macrophages
were found only in mouse models of atherosclerosis, where
3. Macrophage Heterogeneity they were induced by proatherogenic oxidized LDL. Fur-
thermore, proinflammatory macrophages could be induced
One of the key features of macrophages is their high degree of by platelet chemokine CXCL4 [31]. They lose the expression
plasticity that allows them to produce a fine-tuned response of the haemoglobin-haptoglobin scavenger receptor CD163,
to various microenvironmental stimuli [26, 27]. Such plas- which is essential for haemoglobin clearance after the plaque
ticity and heterogeneity made it challenging to achieve a haemorrhage and has therefore protective properties in
comprehensive macrophage classification. Moreover, in vitro atherosclerosis [32].
studies of macrophage activation and differentiation may The described complexity of macrophage phenotypes
not reflect the in vivo situation accurately enough, since urged the development of a more comprehensive classifi-
these processes are fine-tuned by various factors present in cation system to avoid confusion and facilitate interpreta-
the organism’s blood and tissues and can be modelled only tion of data obtained in mice and humans. Joint efforts
roughly. of several experts in the field resulted in formulation of
The identification of pro- and anti-inflammatory macro- guidelines for classification of macrophage phenotypes and
phages led to the establishment of the classical model of polarization pathways [6, 26]. It was recommended to classify
macrophage activation. This model defined two main pheno- the different macrophage phenotypes based on the activa-
types of macrophages: proinflammatory M1 and alternative tion stimulus used and to avoid outdated terminology that
4 BioMed Research International

Table 1: Macrophage phenotypes detected in humans and mice and their role in atherosclerosis (adapted with modifications from [26], with
permission from Elsevier).

Secreted Role in
Phenotype Induction Markers Functions
molecules atherosclerosis
IL-1𝛽, IL-6, IL-6, IL-10 Plaque
IFN-𝛾, TNF-𝛼,
IL-12, IL-23, (low), IL-12 progression,
M1 (human, LPS, and other Th1 response,
TNF-𝛼, CXCL9, (high), IL-23, maintaining
mouse) TRL-mediated antitumor
CXCL10, and TNF-𝛼, iNOS, inflammatory
stimuli
CXCL11 and ROS response
Human: MR,
IL1RN IL-10, TGF-𝛽,
M2a (human, Tissue repair
IL-4, IL-13 Mouse: Arg-1, CCL17, and
mouse) and remodelling
FIZZ1, and CCL22
Ym1/2
IL-6, IL-10 Enriched in
Immune
M2b (human, IL-10 (high), (high), IL-12 regressing plaques
IL-1𝛽, LPS regulatory
mouse) IL-12 (low) (low), and in humans and
functions
TNF-𝛼 mice
IL-10, TGF-𝛽, Phagocytosis,
M2c (human, Human: MR IL-10, TGF-𝛽,
and apoptotic cell
mouse) Mouse: Arg-1 and PTX3
glucocorticoids clearance
TLR + A2 R IL-12 (low), IL-10, VEGF, Present in murine
M2d (mouse) Angiogenesis
ligands TNF-𝛼 (low) and iNOS plaques
Minimal foam cell
MR, MMP7, and IL-6, TNF-𝛼, Weak formation,
M4 (human) CXCL4
S100A8 and MMP12 phagocytosis potentially
proatherogenic
HMOX-1, Nrf2,
Weak Proatherogenic
Mox Oxidized LDL Srxn1, and IL-1𝛽, IL-10
phagocytosis properties in mice
Txnrd1
HA-mac Haemoglobin/ CD163 (high),
HMOX-1 Atheroprotective
(human) haptoglobin HLA-DR (low) Haemoglobin
ABCA1, clearance
M (Hb) Haemoglobin/ Cholesterol efflux,
MR, CD163 ABCG1, and
(human) haptoglobin atheroprotective
LXR𝛼
Mhem (human, Erythrocyte
Heme ATF1, CD163 LXR𝛽 Atheroprotective
mouse) phagocytosis

could lead to confusion. It is currently unclear whether macrophage activation stimuli and proposed a model of
the results of in vitro experiments employing macrophage human macrophage plasticity in inflammatory conditions
activation accurately reflect processes taking place in vivo, defined by transcriptional regulation. Further study of the
since macrophage activation may possibly be induced or genetic mechanisms controlling macrophage activation may
modulated by macrophage isolation procedures. Moreover, result in defining novel therapeutic targets for specific mod-
the results obtained on experimental animals in many ulation of macrophage activation in pathological condi-
cases cannot be directly translated to humans, because the tions.
macrophage subtypes detected in different species (such as
humans and mice) do not fully coincide. As macrophage
activation is dependent on the expression of certain genes, 4. The Role of Different Macrophage
studying changes of gene transcription in response to dif- Types in Atherosclerosis
ferent stimuli will improve our understanding of this pro-
cess. One of the important tools is the recently developed Atherosclerotic lesion site provides a specific microenviron-
transcriptome analysis, which allows studying the complexity ment, enriched with activated cells, modified lipoproteins,
of macrophage activation variations [33]. A recent study and proinflammatory factors, as well as with dying and
has identified a network of transcriptional and epigenetic apoptotic cells. Correspondingly, the macrophage popula-
regulators that orchestrate the activation of proinflamma- tion of atherosclerotic plaques is heterogeneous [35]. The
tory macrophages [34]. The authors analyzed a variety of presence of relatively large numbers of proinflammatory
BioMed Research International 5

macrophages (corresponding to M1 type) in atheroscle- monocytes can have unequal ability to polarize into dif-
rotic lesions is well known [28, 36]. However, alterna- ferent macrophage phenotypes, which can be relevant for
tively activated macrophages have also been detected in the atherosclerosis initiation and progression. It is important to
plaques [37]. Atherosclerotic plaque progression is associated evaluate susceptibility of circulating monocytes to pro- or
with an increase of both macrophages populations, with anti-inflammatory polarization. For that purpose, monocytes
cells expressing proinflammatory markers preferentially dis- were isolated from whole blood of healthy donors, appar-
tributed in shoulder regions that are more susceptible to ently healthy subjects with predisposition to atherosclerosis,
rupture and cells bearing markers of alternative activation and patients with subclinical atherosclerosis evaluated by
located in the adventitia [38]. It has been demonstrated high-resolution ultrasonography of carotid arteries. Mag-
that anti-inflammatory, alternatively activated macrophages netic CD14-positive microbeads were used to obtain a pure
are present in more stable regions of plaques and are more monocyte population. Cells were stimulated with proinflam-
resistant to foam cell formation [39]. Therefore, the pro- matory (IFN-𝛾) or anti-inflammatory (IL-4) factors [44].
and anti-inflammatory macrophage subtypes may reflect the In this simplified experimental model, the production of
plaque progression/instability or regression correspondingly. TNF-𝛼 and CCL18 was used as marker of pro- and anti-
Identification of different types of macrophages in human inflammatory activity, respectively, corresponding to M1 and
tissues remains challenging because of the lack of spe- M2 polarization of macrophages defined by the old paradigm.
cific and reliable markers. Immunohistochemical analysis of This approach revealed a remarkable individual difference
human aorta demonstrated the presence of proinflammatory in monocyte predisposition to activation [45]. This diversity
macrophage marker TNF-𝛼 in atherosclerotic lesions as well did not, however, correlate with the presence or absence of
as in grossly normal areas [40]. However, the quantity of subclinical atherosclerosis in the study subjects.
TNF-𝛼 was increased in the lesion sites, which was also Transcriptome analysis is a powerful modern tool for
confirmed by quantitative PCR analysis of TNF-𝛼 expression. studying monocyte/macrophage activation and function
At the same time, atherosclerotic lesion areas also contained [23]. It provides a set of data on specific genes involved in
cells expressing CCL18, which are likely to be alterna- different stages of macrophage activation. A detailed analysis
tively activated (M2-like) macrophages. More insight into of macrophage activation performed recently [33] explored
macrophage polarization in proatherosclerotic conditions changes in gene transcription induced by 28 different stimuli
was gained by studying macrophage gene expression in vitro. or their combinations. The study identified 49 sets of genes
Incubation of human monocyte-derived macrophages with with similar transcriptional induction that become activated
multiply modified atherogenic LDL resulted in a significant in macrophages in response to various stimuli and specific
increase of intracellular cholesterol accumulation associated transcription factors that regulate them. More studies are
with increased TNF-𝛼 and CCL18 expression [26]. needed, however, to reach an understanding of the complex
Apart from the typical pro- and anti-inflammatory mechanisms of macrophage activation in vivo [46]. It is likely
macrophages that can be classified into M1 and M2 types that macrophage response to various stimuli in different indi-
according to the old activation model, human atheroscle- viduals is largely influenced by genetic variation, especially
rotic lesions contain specific macrophage phenotypes with in genomic regulatory elements that orchestrate the induced
pro- or antiatherogenic properties (Table 1). For instance, activation of macrophage genes. Such influence has recently
CD163-expressing macrophages could be found in haem- been demonstrated, for instance, on F1 crosses of inbred
orrhagic human plaques [41]. These cells are respon- mouse strains [47]. Memory of the past stimuli can also have
sible for haemoglobin clearance and play a protective a profound influence on monocyte ability for activation, as
role in atherosclerotic lesions. Another atheroprotective it has been demonstrated that some stimuli are not easily
macrophage subtype present in humans is Mhem. These cells reversible and can influence the response of the immune
also express CD163, as well as heme-dependent activating system to subsequent stimulation [48].
transcription factor 1 (ATF1), which induces expression of A recent study has revealed an association of mito-
heme oxygenase 1 and liver X receptor- (LXR-) 𝛽. Mhem chondrial gene mutations with monocyte susceptibility to
macrophages participate in haemoglobin clearance via ery- activation [49]. At least three heteroplasmic mutations of
throcyte phagocytosis and have increased cholesterol efflux mtDNA, G14459A, A1555G, and G12315A, associated with
due to expression of LXR-𝛽-dependent genes LXR-𝛼 and atherosclerosis development in humans correlated with facil-
ATP-binding cassette transporter 1 (ABCA1) [42]. These cells itated proinflammatory activation of circulating monocytes.
produce anti-inflammatory IL-10 and apolipoprotein E [43]. Also, two homoplasmic mutations, A1811G and G9477A,
Recently described M4 macrophages can have proathero- tended to correlate with the degree of monocyte susceptibility
genic properties and play a role in the formation of unstable to activation. On the other hand, G9477A mutation inversely
plaques by producing MMP12 and promoting destabilization correlated with the ability of monocytes to become acti-
of the plaque fibrous cap [28]. vated. It is possible that mitochondrial dysfunction caused
by mtDNA mutations activates autophagic clearance and
5. Individual Difference in Macrophage contributed to the development of chronic inflammatory
state, which also plays a role in the development of atheroscle-
Activation and Transcriptome Analysis
rosis. More studies are needed to evaluate the significance
As mentioned above, human macrophages are character- of mitochondrial genome for monocyte/macrophage system
ized by great phenotypic diversity. Moreover, circulating function.
6 BioMed Research International

6. Influence of Lipids on proinflammatory activity of different cholesteryl esters can

Macrophage Activation be conveyed by different signalling pathways; for instance,
7-ketocholesteryl-9-carboxynonanoate was demonstrated to
LDL serves as the primary source of lipid accumulation in activate NF-𝜅B pathway [60] and cholesteryl linoleate-MAP
the arterial wall during atherosclerotic lesion development. In kinase signalling [61]. Another proinflammatory class of
vitro studies have demonstrated that intracellular cholesterol cholesterol derivatives present in atherosclerotic plaques is
accumulation is caused not by native but by atherogenic mod- oxysterol. In macrophages, oxysterol can induce the expres-
ified LDL. Unlike native LDL, modified LDL particles follow sion of proinflammatory monocyte chemoattractant-1 (MCP-
a different metabolic pathway, being internalized mostly 1) [62] and scavenger receptor CD36 [63]. CD36 expression
through unregulated phagocytosis. Macrophages, with their is also stimulated by oxidized cholesterol esters [64]. This
well-developed phagocytic apparatus, are likely to play the scavenger receptor has an important role in atherogenesis, as
key role in this process [28]. its downregulation through stimulation of 𝛼M𝛽2 integrins
Both native and modified LDL were demonstrated to prevented the formation of proinflammatory macrophages
promote proinflammatory polarization of macrophages. A and foam cells [65].
recent study on monocyte-derived macrophages has shown Phospholipase-mediated hydrolysis of lipoproteins
that incubation with LDL resulted in the increased expres- resulting in the release of free phospholipids and fatty
sion of proinflammatory molecules TNF-𝛼 and IL-6 and acids can occur in the acidic plaque microenvironment.
decreased expression of CD206 and CD200R that are typ- These products greatly contribute to lipid accumulation
ical for anti-inflammatory (M2) macrophages [50]. Known in the arterial wall and plaque progression. It has been
forms of modified LDL also have a potent influence on demonstrated that phospholipase A2-treated LDL increased
macrophages promoting the formation of proinflammatory the secretion of proinflammatory TNF-𝛼 and IL-6 by macro-
phenotype. Macrophages recognize modified LDL by means phages and stimulated foam cell formation [66]. The pro-
of TLRs and scavenger receptors. For instance, CD36, a inflammatory signalling of phospholipids and fatty acids is
scavenger receptor, can recognize oxidized LDL and associate mediated by G-protein-coupled receptor G2A, which has an
with TLRs triggering proinflammatory signalling [51]. This important role in the disease pathogenesis, as its deficiency
favours macrophage polarization towards the proinflam- results in advanced atherosclerosis and acquisition of pro-
matory phenotype. TLR activation is accompanied by the inflammatory M1 phenotype by macrophages [67]. Saturated
upregulation of protein kinases C and Syk, activation of fatty acids promote the proinflammatory phenotypic switch
NADPH oxidase 2 (gp91/Nox2), and increased ROS produc- of macrophages through TLR-NF-𝜅B signalling [68].
tion [52]. As a result, macrophages increase the production of Polyunsaturated fatty acids (PUFA) have well-known
proinflammatory cytokines, including IL-1𝛽, and chemokine protective properties in atherosclerosis, which is partly
(C-C motif) ligand 5 (CCL5). Moreover, oxidized LDL can
explained by their anti-inflammatory effects on macrophages.
induce inflammasome activation through CD36 signalling
Conjugated linoleic acid reduced the expression of proin-
[53]. Exposure to oxidized LDL can also promote alternatively
flammatory genes such as NF-𝜅B, CCL2, MMP9, phospho-
activated macrophages to shift their phenotype to a proin-
lipase 2, and cyclooxygenase 2 in macrophages through
flammatory one through altered expression of pro- and anti-
inflammatory genes [54]. peroxisome proliferator-activated receptor 𝛾 (PPAR𝛾) and
It should be noted that the relationship between lipid inhibited atherosclerosis progression in mice. PUFA can
accumulation and proinflammatory activation of macro- also counteract the proatherosclerotic effects of saturated
phages is not straightforward. Lipidomic and transcriptomic fatty acids, such as palmitate-induced expression of lectin-
study conducted in mice fed with normal or high cholesterol like oxidized LDL receptor 1 (LOX1) and fatty acid-binding
high fat diet demonstrated that macrophage-derived foam protein [69]. Eicosapentaenoic acid and dihydroascorbic acid
cells had a “deactivated” phenotype with reduced expression (DHA) have protective effects in atherosclerosis by allevi-
of proinflammatory factors [55]. Such anti-inflammatory ating proinflammatory activity and improving functions of
response was attributed to cellular accumulation of desmos- macrophages [70]. Nitro-fatty acids (NFA) can be formed by
terol, one of the intermediates of cholesterol biosynthesis. interaction of reactive nitrogen species with fatty acids during
In mice, where a specific Mox type of macrophages has oxidative stress [71]. It has been demonstrated that NFA
been described, oxidized phospholipids can induce both pro- possess anti-inflammatory and atheroprotective properties
and anti-inflammatory macrophage phenotypes to transform mediated by Nrf2 and PPAR𝛾 signalling [72]. Attenuation
into Mox through activation of Nrf2, which promotes expres- of atherosclerosis and plaque stabilization due to increased
sion of a number of antioxidant genes [56]. Although Nrf2 collagen deposition was observed in Apoe−/− mice treated
signalling has some protective properties in atherosclerosis, with NFA [73].
its upregulation leads to inflammasome activation, which High-density lipoprotein (HDL) has atheroprotective
renders the Mox switch of macrophages proatherogenic [57]. functions stimulating cholesterol efflux and catabolism [74].
Inflammasome activation in macrophages can result from Decreased relative levels of HDL versus LDL are observed in
phagocytosis of cholesterol crystals that can damage the lyso- atherosclerotic patients. The protective effect of HDL is partly
somal system [58]. Cholesteryl esters that are present in the mediated by its anti-inflammatory activity: normalization of
plaque lipid core can stimulate macrophages and promote the HDL serum levels in atherosclerotic mice led to a decrease
inflammatory response and foam cell formation [52, 59]. The of proinflammatory macrophage numbers in the lesions and
BioMed Research International 7

to an increase of M2 macrophage markers CD163, Arg-

1, and transcription factor FIZZ1 [75]. The expression of
Arg-1 and FIZZ1 was dependent on STAT6 [76]. Another
study has demonstrated that HDL inhibited the proinflam-
matory polarization of macrophages as assessed by such
marker genes as TNF-𝛼, IL-6, and CCL2, as well as surface
markers, but did not stimulate the alternative activation of
macrophages towards the anti-inflammatory phenotype [77].
Modulation of pro- and anti-inflammatory phenotypes of
macrophages by lipids can be considered as a potential point
of therapeutic intervention for treatment of atherosclerosis. Figure 2: The presence of modified LDL, labelled with gold particles
(arrows), in lysosomes of macrophages, visualized in an in vitro
experiment. Transmission Electron Microscopy (TEM). Scale bar =
7. Foam Cell Formation 600 nm.
Intracellular accumulation of lipids is one of the early
events in atherosclerosis development. Foam cell formation
from macrophages is associated with downregulation of and fatty acids, and free cholesterol is trafficked to the
the expression of LDL receptor, which allows these cells endoplasmic reticulum (ER), where it is reesterified by acetyl-
to internalize apoB-containing lipoproteins. Modified LDL, coenzyme A:cholesterol acetyltransferase 1 (ACAT1) [85, 86].
which is internalized by alternative mechanisms, is the Excessive cholesterol uptake has deleterious effects on cells.
primary source of cholesterol accumulation in foam cells, Cholesterol accumulation in the ER membranes leads to
as demonstrated by in vitro studies [78, 79]. Oxidation is its defective esterification by ACAT1 and further increased
the most studied atherogenic modification of LDL. It has storage. ER stress associated with cholesterol storage in
been suggested that increased oxidative stress may account macrophages also contributes to the disease progression
for the formation of atherogenic oxidized LDL and that increasing apoptosis in progressing plaques [87]. Increased
the modified particle can trigger the development of the cell death and impaired clearance of dying cells result in
immune response and induce lipid accumulation in the arte- the formation of necrotic core in advanced atherosclerotic
rial wall [80]. Studying of LDL composition of blood plasma plaques. Cholesterol-rich membrane microdomains facilitate
of atherosclerotic patients revealed different types of LDL proinflammatory TLR- and NF-𝜅B-mediated signalling [88].
modification, including desialylation, glycation, acquisition It should be noted that LDL circulating in the blood of
of negative electric charge, and complex formation [81]. healthy individuals usually does not cause accumulation of
Complex formation renders modified LDL particles espe- lipids in cultured macrophages, whereas LDL of atheroscle-
cially atherogenic. After penetration into the subendothelial rotic patients is in most cases a potent inducer of cellu-
layer of the arterial wall, modified LDL can associate with lar lipidosis. Thus, LDL of atherosclerotic patients, unlike
proteoglycan molecules, which increases its residence time LDL of healthy individuals, is atherogenic. When added to
and promotes lipid accumulation in the arterial wall cells. It primary culture of human monocyte-derived macrophages,
is likely that a complex process of multiple LDL modification atherogenic LDL isolated from the blood of atherosclerotic
occurs in human bloodstream and in the arterial wall. patients induces upregulation of proinflammatory cytokine
Modified LDL can be recognized by macrophages by TNF-𝛼 and anti-inflammatory chemokine CCL18 at the tran-
means of scavenger receptors that play an important role scription level (Table 2). At the same time, nonatherogenic
in atherosclerosis development [82]. Scavenger receptors (native) LDL from healthy individuals had no effect on gene
of macrophages include SR-A1, macrophage receptor with expression when added to cultured macrophages (Table 2).
collagenous structure (MARCO, or SR-A2), CD36, SR- Therefore, multiply modified atherogenic LDL causes pro-
B1, LOX1, scavenger receptor expressed by endothelial and anti-inflammatory macrophage activation. This is a very
cells 1 (SREC1), SR-PSOX, or CXCL16, recognizing phos- important observation considering the significant role of the
phatidylserine and oxidized LDL [5]. In vitro studies have innate immunity and chronic inflammation in the occurrence
shown that degradation of modified (acetylated or oxidized) and development of atherosclerotic lesions. The findings are
LDL by macrophages is mediated mostly by SR-A1 and in good agreement with the results of in situ studies that have
CD36 [78]. Deficiency of these receptors partly inhibited demonstrated upregulation of the expression of pro- and anti-
foam cell formation in Apoe−/− mice, suggesting that other inflammatory cytokines in atherosclerosis [35, 89].
mechanisms of LDL uptake exist in macrophages [83]. Large To assess the impact of modified LDL-induced cholesterol
quantities of native LDL that can be observed in hyperlipi- accumulation on gene expression in macrophages, tran-
demic conditions of growing plaques can also contribute to scriptome study of macrophages incubated with oxidized,
foam cell formation being internalized via pinocytosis [84]. acetylated, and desialylated LDL was performed. Naturally,
Ultrastructural analysis of macrophages incubated with the addition of modified LDL caused changes in the activity of
modified LDL in vitro experiments showed the accumulation hundreds of macrophage genes. It is important to identify the
of LDL in the lysosomes (Figures 2 and 3). Biochemical genes that are associated with lipid accumulation. Incubation
studies revealed that, after internalization, LDL particles are with modified LDL altered the activity of forty genes encod-
degraded in the lysosomal compartments to free cholesterol ing molecules with known functions (Table 3). It should be
8 BioMed Research International

L L
L

Ly
N

Ly
Ly
L

(a) (b)

Figure 3: Foam cells of macrophage origin in an atherosclerotic lesion of the human aorta (a, b). (a) CD68+ cells (brown), some of which
display a typical foam cell appearance (arrows). Immunohistochemistry; peroxidase-anti-peroxidase (PAP) technique; counterstain with
Mayer’s hematoxylin. (b) A large number of lipid inclusions (“lipid droplets”) (L) that fill practically all the cytoplasm in a foam cell in a
human atherosclerotic plaque. Ly: lysosome; N: nucleus. TEM. Scale bars = 100 𝜇m (a) and 2 𝜇m (b).

Table 2: Effect of LDL on cytokine gene expression. lipophagy, which is a special type of autophagy [93]. Studies
on atherosclerosis mouse model demonstrated the protective
Atherogenic role of autophagy through regulation of inflammation [94]
Native LDL
LDL
and cell death [95] in atherosclerotic plaques.
2.0 ± 0.5 (2.1)
TNF-𝛼 1.0 ± 0.3 (1.11)
𝑃 = 0.05
4.4 ± 0.9 (2.8) 8. Macrophage-Based Tests for
CCL18 1.1 ± 0.5 (1.0)
𝑃 = 0.03 Diagnostics and Search of
Monocytes were isolated from whole blood of healthy donors by density Antiatherosclerotic Substances
gradient followed by selection of CD14+ cells by magnetic separation.
Cells were cultured for 7 days. Native or atherogenic LDL was added at Given the crucial role that macrophages play in the partheno-
a concentration of 100 𝜇g/mL and the cells were incubated for 24 hours.
RNA was isolated and gene expression was measured by RT-PCR technique.
genesis of atherosclerosis, it is important to establish reliable
The table shows the relative expression of the genes. As 1, the control gene monocyte/macrophage-based models that can be used for
expression (without LDL) was taken. Values in parentheses are standard studying molecular mechanisms of the disease pathogen-
deviations. esis as well as for screening of potential antiatheroscle-
rotic substances. Recently, a monocyte/macrophage-based
noted that most of these genes (26 of 40) may be related assay was developed to evaluate the changes in patient’s
to innate immunity function. This observation suggests that monocyte response to pro- and anti-inflammatory stimuli,
LDL-induced cholesterol accumulation in macrophages trig- which would reveal the possible bias of the macrophages
gers an immune response. Further research should explain polarization towards M1 or M2 phenotype. A pure population
the link between the intracellular lipid accumulation and of monocytes/macrophages was obtained using magnetic
chronic inflammation in atherosclerotic lesions. separation method [96]. Isolated cells were stimulated with
Increased lipid efflux could be a powerful therapeutic proinflammatory LPS and IFN-𝛾 or with anti-inflammatory
option for treatment of atherosclerosis. Several proteins IL-4 [97]. Macrophage polarization was assessed by measur-
facilitate lipid efflux in macrophages, including ABCA1 and ing the production of pro- and anti-inflammatory cytokines
ABCG1 [90]. ABCA1 and ABCG1 mediate lipid efflux to by ELISA. Proinflammatory activity of macrophages was
HDL particles and are upregulated in response to increased assessed by the levels of secreted TNF𝛼 and IL-1𝛽, and anti-
cellular cholesterol levels sensed by liver X receptors (LXRs). inflammatory activity was assessed by the levels of CCL18
Their activation has also anti-inflammatory effects [91]. It was and IL-1Ra. Inflammasome activation can also be assessed
demonstrated that LXR activation in murine macrophages in this system by measuring the IL-1𝛽 expression at mRNA
lacking ABCA1/G1 had a strong antiatherosclerotic effect, level and comparing the results with the amount of mature IL-
decreasing lesion area and complexity through reduction of 1𝛽 detected by ELISA or by measurement of active caspase-1,
inflammation [92]. The therapeutic option of activating LXRs TNF-𝛼, and IL-8 [98]. Characterization of macrophages can
for treatment of atherosclerosis is currently being explored. be performed by the analysis of a panel of markers, including
Another mechanism of cholesterol clearance from cells is MMR, CD163, TGF-RII, CSFR1, TNFRI, CD16, CD32, CD64,
BioMed Research International 9

Table 3: List of macrophage genes whose activity changes in the accumulation of intracellular cholesterol.

Gene Molecule Functions

FCGBP Fc fragment of IgG binding protein Immune response
S100A8 S100 calcium binding protein A8 Immune response, migration, cell body formation
ITLN1 Intelectin 1 (galactofuranose binding) Pathogen metabolism
NCOR2 Nuclear receptor corepressor 2 Immune response
TPPP3 Tubulin polymerization-promoting protein family
Cell body formation
member 3
AKR1C1 Aldo-keto reductase family 1, member C1 Immune response
FAM65A Family with sequence similarity 65, member A Cell body formation
HECTD2 HECT domain containing E3 ubiquitin protein ligase 2 Metabolism
RD3 Retinal degeneration 3 Nerve features
TNFSF18 Tumor necrosis factor (ligand) superfamily, member 18 Immune response, migration
NEURL3 Neuralized E3 ubiquitin protein ligase 3 Metabolism
CD209 CD209 molecule Immune response, migration, dendritic cell features
STRIP2 Striatin interacting protein 2 Cell body formation
CCL4L2 Chemokine (C-C motif) ligand 4-like 2 Migration
TJP2 Tight junction protein 2 Migration
SPON2 Spondin 2, extracellular matrix protein Migration
L1CAM L1 cell adhesion molecule Migration
ARHGEF16 Rho guanine nucleotide exchange factor (GEF) 16 Migration
NES Nestin Cell body formation, nerve features
F3 Coagulation factor III (thromboplastin, tissue factor) Migration
GALNT5 Polypeptide N-acetylgalactosaminyltransferase 5 Metabolism
MT1E Metallothionein 1E Metabolism
COQ2 Coenzyme Q2 4-hydroxybenzoate
Metabolism
polyprenyltransferase
TRIM54 Tripartite motif containing 54 Cell body formation
ANKRD63 Ankyrin repeat domain 63 Cell body formation
CCL24 Chemokine (C-C motif) ligand 24 Immune response, migration
HIVEP3 Human immunodeficiency virus type I enhancer
Immune response
binding protein 3
NETO2 Neuropilin (NRP) and tolloid- (TLL-) like 2 Nerve features
CCL4 Chemokine (C-C motif) ligand 4 Immune response, migration
ACPP Acid phosphatase, prostate Metabolism
STARD4 StAR-related lipid transfer (START) domain containing
Metabolism
4
RANBP10 RAN binding protein 10 Cell body formation
ROBO2 Roundabout guidance receptor 2 Migration, nerve features
CHL1 Cell adhesion molecule L1-like Migration, nerve features
Negative regulation of interferon-gamma production;
RARA Retinoic acid receptor, alpha positive regulation of interleukin-4 production,
immune response
SLC16A9 Solute carrier family 16, member 9 Metabolism
HTR2A 5-Hydroxytryptamine (serotonin) receptor 2A,
Nerve features
G-protein-coupled
BCAR1 Breast cancer antiestrogen resistance 1 Migration
OR6K3 Olfactory receptor, family 6, subfamily K, member 3 Nerve features
CYP7B1 Cytochrome P450, family 7, subfamily B, polypeptide 1 Metabolism
10 BioMed Research International

and stabilin-1, as well as expression of TLR1, TLR2, and TLR4 types of macrophages in atherosclerosis progression and on
at mRNA level and on the cell surface. development of macrophage-targeting therapies.
Monocyte/macrophage-based method was used to ana-
lyze activation of monocytes isolated from blood of healthy Competing Interests
subjects (𝑛 = 19), atherosclerosis patients (𝑛 = 22), and
breast cancer patients (𝑛 = 18). It was demonstrated that The authors report no competing interests regarding the
the production of proinflammatory TNF-𝛼 was significantly publication of this paper.
lower in atherosclerotic patients and significantly higher in
cancer patients in comparison to healthy subjects, whereas
Acknowledgments
the production of anti-inflammatory CCL18 was decreased in
both atherosclerosis and cancer patients [40]. Comparison of This work was supported by the Russian Science Foundation
subjects with predisposition to atherosclerosis (𝑛 = 21, mean (Grant no. 14-15-00112).
age 63±9 years), subjects with subclinical atherosclerosis (𝑛 =
21, mean age 62±7 years), and healthy subjects (𝑛 = 21, mean
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