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Atherosclerosis and its complications represent the leading death cause worldwide, despite many therapeutic developments. Atherosclerosis is a complex, multi-stage disease whereby perturbed lipid metabolism leads to cholesterol accumulation into the vascular walls and plaque formation. Generation of apoE-/- and LDLR-/- atherosclerosis mouse models opened the avenue for investigating the mechanisms of action for specific molecules. We focus herein on the involvement of non-lipoprotein receptors in atherogenesis, as revealed by their total or site-specific ablation in the aforementioned murine models. The receptors reviewed span a broad range, from molecules related to lipid metabolism (adiponectin receptors) to molecules whose connection with atherogenesis is less obvious (cannabinoid receptors). We also outline cross-transplantation studies which allowed uncoupling the lipid modulating effects from the inflammatory ones. For certain receptors, since knockouts were unavailable, pharmacological data are presented instead. We emphasize the contribution of the receptors to the pathology, based on functional criteria, such as oxidative stress, immune response, inflammation, angiogenesis. Controversial aspects regarding the pro- or anti- atherogenic activity of some receptors are highlighted. We assume these discrepancies are due to the experimental setup, animal models used, tissue specific action, various isoforms analyzed, divergent signaling or cross-talk between metabolic and immune pathways. Understanding the influences of cellular receptors in the progression of atherosclerosis allows their modulation toward an anti-atherogenic phenotype. The experimental studies in animal models were in some cases successfully extrapolated to humans leading to atheroma reduction, and we expect this to occur even to a greater extent, based on the newest achievements.
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Current Molecular Medicine 2015, 15, 1-27 1
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Beyond Lipoprotein Receptors: Learning from Receptor Knockouts
Mouse Models about New Targets for Reduction of the
Atherosclerotic Plaque
V.G. Trusca, E.V. Fuior and A.V. Gafencu*
Department of Genomics, Transcriptomics and Molecular Therapies, Institute of Cellular
Biology and Pathology “Nicolae Simionescu”, Bucharest, Romania
Abstract: Atherosclerosis and its complications represent the leading death cause
worldwide, despite many therapeutic developments. Atherosclerosis is a complex, multi-
stage disease whereby perturbed lipid metabolism leads to cholesterol accumulation into the
vascular walls and plaque formation. Generation of apoE-/- and LDLR-/- atherosclerosis
mouse models opened the avenue for investigating the mechanisms of action for specific
molecules. We focus herein on the involvement of non-lipoprotein receptors in
atherogenesis, as revealed by their total or site-specific ablation in the aforementioned
murine models. The receptors reviewed span a broad range, from molecules related to lipid metabolism
(adiponectin receptors) to molecules whose connection with atherogenesis is less obvious (cannabinoid
receptors). We also outline cross-transplantation studies which allowed uncoupling the lipid modulating effects
from the inflammatory ones. For certain receptors, since knockouts were unavailable, pharmacological data
are presented instead. We emphasize the contribution of the receptors to the pathology, based on functional
criteria, such as oxidative stress, immune response, inflammation, angiogenesis. Controversial aspects
regarding the pro- or anti- atherogenic activity of some receptors are highlighted. We assume these
discrepancies are due to the experimental setup, animal models used, tissue-specific action, various isoforms
analyzed, divergent signaling or cross-talk between metabolic and immune pathways. Understanding the
influences of cellular receptors in the progression of atherosclerosis allows their modulation towards an anti-
atherogenic phenotype. The experimental studies in animal models were in some cases successfully
extrapolated to humans leading to atheroma reduction, and we expect this to occur even to a greater extent,
based on the newest achievements.
Keywords: Atherosclerosis, membrane receptors, mouse models, atherosclerotic plaque, endothelial dysfunction,
macrophages, vascular smooth muscle cells.
Atherosclerosis is a complex disease in which lipid
metabolism is perturbed, leading to cholesterol
accumulation into the vascular walls and plaque
formation [1]. In the past decade, the contribution of
inflammation and immune responses in the progression
of the atherogenesis was highlighted [2]. Nowadays,
atherosclerosis is considered a multifactorial disease
and thus, its treatment has to include a variety of
To identify potential therapeutic targets for the
prevention or treatment of atherosclerosis, different
approaches have been developed. The anti-
atherosclerotic strategies were designed to increase
the level of the atheroprotective proteins as well as to
decrease or abrogate the gene expression of the pro-
atherogenic proteins. The targets represented by the
*Address correspondence to this author at the Department of
Molecular Biology and Pharmacology, Institute of Cellular Biology
and Pathology, “Nicolae Simionescu”, 8, B. P. Hasdeu Street, Sect 5,
Bucharest-050568, Romania; Tel: (4021) 3194518;
Fax: (4021) 3194519; E-mail:
molecules involved in the lipid metabolism, such as
apolipoproteins (apoB, apoAI, apoE, apoCIII, etc.),
transporters (ABCA1, ABCG1), receptors (CD36, SRBI,
LDLR, etc.), enzymes (ACAT, LCAT, CETP, etc.)
paved the way for therapeutic efforts to attenuate
atherosclerosis. Discussion of these targets is outside
the scope of our review, since there are numerous
studies that have already addressed them [3-7]. All
these molecules play pivotal roles in maintaining the
proper balance of lipid metabolism, but in the
pathological states, their deregulated expression,
improper activity or their incapacity to metabolize high
amounts of dietary lipids lead to increased lipid
deposition in the atherosclerotic plaques. In addition to
the proteins involved in lipid metabolism, other proteins
involved in cellular and molecular processes that take
place in atherogenesis, leading to oxidative stress,
inflammation (adhesion and infiltration of monocytes in
atherosclerotic plaque) or angiogenesis may represent
valuable targets in order to reduce or attenuate the
atherosclerotic plaques.
A variety of small and large animal atherosclerosis
models were used to investigate the events occurring
A.V. Gafencu
2 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
in the arterial wall during atherogenesis, to obtain
valuable information about molecular markers for
diagnostic and to assess the potential of the thera-
peutic strategies for this disease. The most commonly
used murine model is apoE-deficient (apoE-/-) mouse
that develops extensive atherosclerotic lesions similar
in structure with human atheromas, even on the chow
diet. However, administration of Western (high fat) diet
accelerates the development of the atherosclerotic
plaques at all stages and the lesions are more lipid-rich
than those of animals fed with the chow diet [8]. The
major disadvantage of using apoE-/- mice is that the
plasma cholesterol is mostly carried on lipoprotein
remnants (containing apoB48 as the main apoB
protein) rather than on LDL, as in humans. LDLR-
deficient (LDLR-/-) mice were generated by Ishibashi et
al. and represent a model of familial hypercholesterolemia
[9]. An advantage of LDLR-/- mice is that LDL-receptor
is not involved in a multitude of functions as described
for apoE. The LDLR-/- mice have elevated LDL levels
and develop small lesions on the chow diet, but the
severity of the hypercholesterolemia and the
atherosclerotic lesions is accelerated when these mice
are fed with the Western-type diet [10]. The plasma
lipoprotein profile of LDLR-/- mice is similar to that of
humans, having cholesterol confined mainly to LDL
fraction. The absence of the LDL-receptor mainly
influences lipoprotein uptake and clearance, resulting
in a greater preponderance of LDL as the cholesterol
carrying plasma lipoprotein. The morphology of the
lesions in the LDLR-/- mice is comparable to that in
apoE-/- mice; however, the LDLR-/- mouse represents a
moderate model in comparison with the apoE-/- mouse,
mainly because of the milder degree of hyperlipidemia
In order to shed some light on the main processes
occurring in atherogenesis and to identify novel
molecules that could have a beneficial impact on this
disease, a multitude of proteins were targeted. The
main classes of potential therapeutic target proteins for
atherosclerosis are: (i) membrane receptors (described
in this review), (ii) adhesion molecules, such as
VCAM1 [12], integrins [13], selectins [14, 15], (iii)
extracellular and intracellular enzymes, such as ACAT
[16], lipoprotein-associated phospholipases [17],
glutathione peroxidases [18], matrix metalloproteinases
[19, 20], endothelial NO-synthase [21], caspases [22],
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)
reductases [23], geranylgeranyltransferases [24] or
sphingomyelin synthases [25], (iv) extracellular
signaling molecules, such as cytokines [26, 27], and
molecules of the intracellular pathways, such as Akt
[28-30], AMPK [31], Jak [32], JNK [33], (v) transcription
factors, such as NF-κB [34], ATF [35], AP-1 [36] or
CHOP [37, 38] and nuclear receptors, such as LXR
[39], RXR [40], FXR [41], PPAR [42], etc.
Here we focus on the membrane receptors that are
not involved in the lipid metabolism, but take part in the
main atherogenic events. Thus, we group these
receptors by their implication in different processes that
lead to atherosclerosis progression, such as oxidative
stress, immune response, inflammation, angiogenesis,
as summarized in Table 1. However, despite that some
of the receptors discussed here are assigned to a
specific category, most of them play multiple roles in
There are two main experimental strategies to
detect the influence of a target receptor on
atherosclerosis in animal models: (i) pharmacological
modulation by the use of agonists or antagonists in an
atherosclerosis mouse model; (ii) generation of double
knockout mice by ablation of a specific gene on the
background of an atherosclerosis model; if the
complete ablation has lethal effect, partial deletion or
conditional expression can be obtained and analyzed.
In Fig. (1), we depicted these receptors on the most
important cell types found in the atheroma plaque
according to their main pro- or anti- atherogenic action
(showed in red, respectively green color, in the figure).
Important findings regarding the therapeutic potential of
receptors involved in the various events
aforementioned are discussed below.
The generation of reactive oxygen species (ROS)
playing a remarkable role in normal cardiovascular
signaling is tightly regulated. However, the oxidative
stress can occur due to an excessive production of
reactive oxygen / nitrogen species or an altered
antioxidant system, promoting atherosclerosis.
The macrophages and smooth muscle cells (SMC)
were described as the main sources of oxidative
molecules and ROS in atherosclerotic vessels [43]. In
addition, plasma lipoproteins, due to their susceptibility
to oxidative modification, can trigger the oxidative injury
of the arterial wall, being recognized as natural
biosensors of the oxidative status of the inflamed
endothelium [44]. The initial hypothesis stating that
antioxidants (which interfere with LDL oxidation and
prevent endothelial dysfunction) could reduce the level
of atherosclerotic lesions was tested in clinical trials.
Unexpectedly, the use of antioxidant vitamins C and E
have shown only minor beneficial effects on
atherosclerosis [45]. Therefore, the identification of
novel therapeutic targets effective in inhibiting oxidative
processes and consequently preventing atherosclero-
sis continues to be a priority. Among the receptors
playing a role in the oxidative stress, in this review we
discuss the atheroprotective or atherogenic function of
the receptors for angiotensin, endothelin and adipo-
Angiotensin Receptors
The role of angiotensin (AT) II in atherogenesis is
well known [46]. To date, two subtypes of angiotensin II
receptors, AT1R and AT2R, exerting antagonistic
activity, have been identified [47]. There is only one
AT1R isoform in humans, while rodents have two AT1R
subtypes, AT1AR and AT1BR [48]. Pharmacological
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 3
Table 1. Membrane receptors used as targets for atherosclerosis in experimental models.
(+) /Atherogenic (-)
Experimental design
Oxidative stress
Mφ-specific AdipoR1LDLR-/- mice.
-/-apoE-/- mice.
AT1AR-/-apoE-/- mice; AT1AR-/-
LDLR-/- mice; Losartan or
candesartan antagonists.
[56, 57, 59]
AT2R-/-apoE-/- mice; AT2R
activation in AT1AR-/-apoE-/-mice.
Endothelin R
ETAR selective antagonists or
ETAR-ETBR non-selective
antagonists in apoE-/- or LDLR-/-
[80, 81, 83, 86,
Immune response and inflammation
FcγR-/-apoE-/- mice expressing only
FcγRIIB; FcγRIIB-/-apoE-/- mice;
LDLR-/- mice transplanted with
FcγRIII-/-LDLR-/- mice.
TLR2-/-apoE-/- mice; TLR2-/-LDLR-/-
mice; TLR2-/-in BMDC; Neutralizing
[125, 126, 327]
(+) or (-)
TLR3-/-apoE-/- mice; TLR3
stimulation in apoE-/- mice.
[131, 133]
TLR4-/-apoE-/- mice; TLR4-/-LDLR-/-
[123, 137]
TLR7 inactivation in apoE-/- mice.
TLR9-/-apoE-/- mice.
LDLR-/- mice transplanted with
BMDC of p55 TNFR-/-mice.
Prostanoid R
ApoE-/-IP-/- mice.
TxA2 R
ApoE-/-TP-/-mice; Treatment of
apoE-/- mice with S18886.
[148, 151-153]
GLP-1R agonists; Liraglutide
Prolactin R
LDLR-/- mice transplanted with
BMDC from transgenic mice
expressing Del1-9-G129R-hPRL.
Leptin R
Ablation of leptin R in apoE-/- mice.
Leptin treatment in Leprs/sapoE-/- or
Leprdb/dbapoE-/- mice.
Nicotinic R
Niacin treatment in GPR109A-/-
LDLR-/- mice; MK-1903 and
SCH900271 agonists; GPR109A
Cannabinoid R
Administration of THC in apoE-/-
mice; CB1R antagonist rimonabant
in LDLR-/-mice.
[200, 211]
4 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
antagonists and genetic studies in mice revealed that
the main physiological and pathological effects of
angiotensin II are mediated by the AT1R [49, 50].
In humans, AT1Rs are widely expressed by most cell
types, while in mice, AT1Rs were found to be
differentially expressed: AT1ARs are expressed in the
(Table 1) contd..
(+) /Atherogenic (-)
Experimental design
CB2R-/-LDLR-/- mice; CB2R-/-apoE-/-
mice; Lack of CB2R in the
hematopoietic cells of LDLR-/-
mice; ApoE-/- mice treated with
[66, 212-214]
Serotonin R
5-HT2A R
Sarpogrelate, a selective 5-HT2A
receptor antagonist.
Tie1+/-apoE-/- mice;
ApoE-/- mice with endothelial-
specific Tie1 deletion.
Blocking Tie2 activation; Tie2
vaccination of LDLR-/- mice.
[257, 258]
Administration of SSR128129E
antagonist in apoE-/- mice.
[265, 266]
Hematopoietic apoE-/-PDGFB-/-
chimeras or apoE-/- mice treated
with CT52923; ApoE-/- mice
injected with APB5 anti-PDGFRβ;
Treatment with imatinib of diabetic
apoE-/- mice.
Haplodeficient TrkB+/-apoE-/- mice.
Other processes
A1AR-/-apoE-/- mice; A1R agonists /
antagonists or siRNA.
[287, 288]
A2aAR-/-apoE-/- mice.
A2bAR-/- apoE-/- mice.
P2Y1R whole body or organ-
targeted ablation in apoE-/- mice.
-/- LDLR-/- mice.
-/-apoE-/- mice.
P2X1 knockout mice or inhibition of
P2X1 with selective antagonists.
Ablation or overexpression of P2X4
in the heart.
Activation of P2X7; P2X7
-/- mice;
P2X7 inhibition using KN6 blocker,
shRNA or siRNA.
[307, 309, 310]
(+) or (-)
Transgenic mice expressing
human S-endoglin in EC.
[316, 318, 319,
Insulin R
Insulin R
Specific IRS-2-/- in Mφ; Systemic
IRS-2-/- in apoE-/- mice;
ApoE-/- mice heterozygous for IR
and IRS-1; LDLR-/- mice
transplanted with BMDC from IR-/-
mice; Fetal liver cell transplantation
of IRS-2-/-apoE-/- cells in apoE-/-
[331, 334, 335]
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 5
kidney, liver, adrenal gland, brain, ovary, testis, lung,
heart, and adipose tissue, whereas AT1BRs were
detected only in the adrenal gland, brain, and testis
[48]. AT2R expression is highest during fetal
development, decreases in most tissues to very low
levels in adults and is up-regulated in many
pathological conditions [51]. Noteworthy, AT1AR
activation represents one of the main sources of
oxidative stress in the vasculature [52]. Large amount
of data indicate that genetic disruption or
pharmacological inhibition of AT1AR ameliorates
atherosclerosis development in both apoE-/- and LDLR-
/- mice [53-55]. The key role of AT1R in diabetes-
induced atherosclerosis was recently revealed, Tiyerili
et al. demonstrating that AT1R inhibition improves
endothelial function and exerts anti-atherosclerotic
effects in part mediated via PPARγ in mice [56].
Fukuda et al. showed that chimeric mice having
disrupted AT1ARs in bone marrow cells displayed
reduced atherosclerosis, demonstrating that AT1ARs
expressed by bone marrow cells promote disease
progression [57]. Losartan, an angiotensin receptor
antagonist, significantly reduced the evolution of
atherosclerosis, and stabilized the plaques of apoE-/-
mice [58]. Recent data showed that lipid and
macrophage accumulation, expression of inflammatory
cytokines as well as MMP-9 activity in atherosclerotic
plaques were significantly suppressed in AT1AR-/-apoE-/-
mice as compared with apoE-/- mice [59]. Total ablation
of AT1ARs in LDL-/- mice drastically decreased
atherosclerosis and aneurysm, while specific deletion
of this receptor in endothelial cells (EC) or SMC had no
significant effect on atherosclerotic lesion size and
aneurysm [60]. Treatment with high-dose of
candesartan, an AT1R blocker, induced regression of
atherosclerosis in apoE-/- mice and Hayashi et al.
suggested that the mechanism involves decreased lipid
retention (through reduction in the lipid-retaining
proteoglycan biglycan) and increased lipid release
(through suppression of ACAT1 expression) [61]. The
aggravation of atherosclerosis in AT2R-/-apoE-/- mice as
compared with control apoE-/- mice was noticed and
modulation of NADPH oxidase activity was identified as
the main mechanism [62]. Several studies reported the
beneficial effects of AT2R stimulation in the vasculature
and recent evidence indicated an important anti-
atherosclerotic role of AT2Rs [63, 64]. Direct AT2R
activation conferred pronounced atheroprotective
effects in AT1A-/- apoE-/-mice, as shown by Tiyerili et al.
[63, 64]. Apart from reducing the oxidative stress, the
inhibition of NF-κB activity is a key molecular
mechanism involved in the AT2R-mediated anti-
inflammatory actions [65].
The effect of receptor-associated protein (RAP) [66]
deficiency in angiotensin II-induced atherosclerosis
was explored in either RAP+/+LDLR-/- or RAP-/-LDLR-/-
mice infused with angiotensin II [67]. RAP was initially
described as a chaperone regulating the expression of
the LDLR-related protein 1 (LRP1), but now it is known
to regulate many proteins [68]. The study of Wang et
al. described a reduction of atherosclerosis in RAP-/-
LDLR-/- mice and no effect on angiotensin II-induced
aneurysm, suggesting that the diversity of aortic SMC
may contribute to these differential effects [67]. In
2010, the anti-atherogenic and vasoprotective effects
of angiotensin-(1-7) receptor Mas in apoE-/- mice were
discovered [69]. In 2012, Jawien et al. reported that the
angiotensin-(1-7) receptor Mas agonist, AVE 0991,
alleviated the atherogenesis in apoE-/- mice by
decreasing NADPH oxidase expression and therefore
reducing ROS production [70]. Shortly after, the
influence of this agonist on mitochondrial dysfunction,
an important feature of cardiovascular and renal injury,
was studied and the results showed that AVE 0991
agonist partially reversed atherosclerosis-related
changes in apoE-/- mice [71]. The generation of Mas-/-
apoE-/- mice allowed to investigate the effects of Mas
receptor deletion which was associated with the
alteration of lipid profile and severe hepatic steatosis
[72]. Noteworthy, Mas receptor deficiency did not
significantly affect atherogenesis or intra-plaque
inflammation in comparison with control apoE-/- mice.
Lately, it has been shown that chronic angiotensin-(1-7)
treatment decreases endothelial dysfunction and
reduces ROS production that in turn causes decreased
activity of p38 mitogen-activated protein kinase [73].
Angiotensin-(1-7) treatment had no effect in Mas-/-
apoE-/- mice confirming the specificity of angiotensin-(1-
7) action through Mas receptor.
Endothelin Receptors
The endothelin family is represented by three active
isoforms: endothelin 1, 2, and 3 [74], the first being the
most important. Endothelin 1 (ET1) is a potent
vasoconstrictor agent, which induces the release of
prostacyclin, vasopresin, inhibitors of renin, endothelial-
derived relaxing factors, and has an antithrombotic role
[75]. Overexpression of ET1 inhibited vascular nitric
oxide production and induced the formation of
atherosclerotic plaques [76]. Among the ET receptors,
ETAR is found mainly on vascular SMC and induces
vasoconstriction and proliferation, while ETBR is found
on endothelial, epithelial, endocrine, and nerve cells
and induces vasorelaxation and the removal of ET1
[77]. As shown by Barton et al., ET1 has pro-
inflammatory activity, causes endothelial dysfunction
and atherosclerosis development in apoE-/- mice
treated with a Western-type diet [78]. Further studies
highlighted that the overexpression of ET1 significantly
increases atherosclerosis in apoE-/- mice fed with a
high-fat diet [79]. Therefore, blocking ET receptors,
particularly ETAR, represents an important step in
preventing the pathological effects of ET1. There is
consistent evidence that selective ETAR inhibition by
administration of LU135252, significantly decreases the
level of atherosclerotic lesions in apoE-/- mice [80] as
well as in hypercolesterolemic LDLR-/- mice [81].
Blocking ETAR by LU135252 administration in apoE-/-
mice had no effect on blood pressure or plasma lipid
profile, but entirely restored NO-mediated endothelium-
dependent relaxation, indicating an increased NO
bioavailability. Significant beneficial effects were also
observed in other atherosclerosis animal models, such
as cholesterol-fed hamsters, where ETAR antagonism
6 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
ameliorated early atherosclerosis [82]. In 2013, Yoon et
al. showed the attenuation of coronary plaque
progression in segments with endothelial dysfunction in
patients with non-obstructive coronary disease or early
coronary atherosclerosis after long-treatment with
atrasentan, a highly selective antagonist of ETAR [83].
Results of trials with ETR antagonists administered in
pulmonary arterial hypertension as well as the effects
of ETR antagonists in inflammatory response, sepsis
and other several disorders were recently discussed
[84, 85]. ETR selective antagonists, such as BQ123
[86] and BQ788 [87], as well as ETAR-ETBR non-
selective antagonists, bosentan [88] and tezosentan
[89], seem to have beneficial effects, but further
investigations are required to clarify their actions in
respect to atherosclerosis.
Adiponectin Receptors
Adiponectin, a metabolically active cytokine
secreted from adipose tissue, signals through the
adiponectin receptors, AdipoR1 and AdipoR2. These
receptors have opposing roles [90] and different
localization: AdipoR1 is well expressed in skeletal
muscle and macrophages, whereas AdipoR2 is
predominantly expressed in the liver [91]. Adiponectin
levels are not correlated with a suppression of the
atherosclerosis process in mice using adiponectin
deficient or overexpressing mice crossed with either
LDLR-/- or apoE-/- mice [92]. The first evidence of the
anti-atherogenic properties of adiponectin was obtained
using apoE-/- mice treated with recombinant adenovirus
expressing human adiponectin [93]. In apoE-/- mice,
adiponectin diminishes atherosclerotic plaques by
inhibiting the inducible NOS and thus, reducing
oxidative/nitrative stress [94].
Recently, Luo et al. generated transgenic mice
which specifically overexpress the gene coding for
AdipoR1 in mouse macrophages using the human
scavenger receptor A-I gene (SR-AI) enhancer/
promoter [95]. Macrophage-specific AdipoR1-/- mice
exhibited reduced whole body weight, fat accumulation
and liver steatosis when fed a high fat diet. Moreover,
macrophage-specific AdipoR1-transgenic animals
crossbred with LDLR-/- mice presented reduced
macrophage foam cell formation in the arterial wall [95].
The effect of AdipoR2- ablation on the progression of
atherosclerosis was evaluated in apoE-/- mice [96].
Data showed that adipoR2-/-apoE-/- mice on athero-
genic diet had decreased plaque area in the brachio-
cephalic artery compared with adipoR2+/+apoE-/- litter-
mate controls. In addition, lower CD36 and higher
ABCA1 mRNA levels as well as decreased accumu-
Fig. (1). Schematic representation of non-lipoprotein receptors, as promising targets for atherosclerosis plaque
regression. The figure illustrates the main targeted receptors expressed on the key cells of the atheromatous plaque:
endothelial cells (EC), macrophages (MΦ), dendritic cells (DC), smooth muscle cells (SMC) and activated platelets (PLT). In
order to simplify the picture, the mainly atheroprotective (green) and predominantly atherogenic receptors (red) are presented
on two different macrophages or grouped on SMC and on the endothelium layer.
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 7
lation of neutral lipids in peritoneal macrophages after
exposure to oxidized LDL were reported [96]. These
results indicate that activating AdipoR1 or antagonizing
AdipoR2 may be an attractive therapeutic approach for
atherosclerosis treatment.
A growing body of evidence indicates that immune
mechanisms and inflammation play a crucial role in
atherogenesis [97, 98]. It is well known that the
immune and inflammatory responses lead to cell
recruitment (mostly macrophages and lymphocytes)
and retention into arterial lesions. Hence, attempts
were made in order to reduce the number of
macrophages (which represent the main source of
foam cells) and therefore to decrease atherosclerotic
lesion without altering blood lipids. Systemic
administration of liposome-encapsulated clodronate
induced inactivation of macrophages which reduced
neointimal hyperplasia in rabbits, rats or hamsters, but
also removed blood monocytes and increased the lipid
content of the lesions [99, 100]. Although it was
expected that this strategy would likely attenuate the
inflammatory response and subsequently improve
plaque stability, adverse effects have been noticed due
to unselective drug availability [101]. Therefore, the
development of additional anti-inflammatory therapies
is of great interest for atherosclerosis.
IgG Receptors
The receptors for the Fc region of IgG (FcγRs) are
broadly expressed on the cells of the hematopoietic
system and play a pivotal role in the immune and
inflammatory responses. Human FcγR family includes
high affinity receptors (FcγRI) and low affinity receptors
(FcγRIIA-C, FcγRIIIA-B). In human atherosclerotic
lesions, the presence of FcγRs was revealed,
suggesting their potential role in atheromatous lesion
formation and the involvement of the immune
complexes in atherogenesis [102-104]. The presence
of IgG in the atherosclerotic plaque was detected a
long time ago [105], but it is not well established how
IgG reaches the atherosclerotic plaque: IgG may be
produced in the atheromatous plaque by the
transmigrated lymphocytes or it can be transcytosed
through activated endothelium that expresses FcγRs.
Considering that IgG may have anti-atherosclerotic
effects, Yuan et al. demonstrated that intravenous
treatment with normal polyclonal immunoglobulins
reduces the atherosclerotic lesions in
apoE-/- mice [103]. This effect may be a result of the
FcγR-mediated immunomodulatory actions.
Macrophage-derived foam cell development implies,
apart from the scavenger receptor pathway, the uptake
of lipoproteins in immune complexes through FcγRs. It
was demonstrated that macrophage-foam cells
development takes place predominantly through FcγRI
and FcγRII [106, 107]. The FcγR-mediated immune
reaction induces a variety of metabolic and functional
changes in macrophages which are likely to contribute,
directly or indirectly, to the endothelial damage and the
accumulation of macrophages in human atherosclerotic
lesions [108, 109]. The double-knockout mice
generated by crossing apoE-/- mice with FcγR chain-
deficient mice, expressing only the inhibitory FcγRIIB
receptor, developed significantly reduced
atherosclerotic lesions compared with apoE-/- mice
[110]. Moreover, FcγRIIB-/-apoE-/- mice developed
intensified atherosclerosis, accompanied by increased
aortic antibody levels to pro-inflammatory cytokines
and modified LDL [111]. Zhao et al. evaluated the
FcγRIIB role in atherosclerosis in LDLR-/- mice
transplanted with FcγRIIB-/- bone marrow cells. They
noticed that the plaques in the descending aorta were
significantly larger in mice transplanted with FcγRIIB-/-
bone marrow cells than in mice transplanted with
normal bone marrow cells, but the plasma cholesterol
or triglyceride concentrations were similar in the two
groups [112]. Recent data showed that FcγRIIB-/-apoE-/-
mice had reduced atherosclerosis and their plaques
displayed a systemic anti-inflammatory phenotype
associated with higher expression of arginase 1 and
lower expression of inducible NOS [113].
In FcγRIII-/-LDLR-/- mice, the arterial lesion formation
was dramatically decreased in advanced stages (but
not in early stages) of atherogenesis compared with
LDLR-/- mice. This reduction was associated with an
increased production of IL-10 through an expansion of
CD4+T-cells and with upregulated IgG1 and IgG2c
titers to oxidized LDL [114].
Endothelial cells do not express FcγRs in normal
conditions. The expression of functional FcγRs was
found on EC incubated with TNFα, interferon γ or with
hyperlipemic serum from patients with cardiovascular
diseases [115]. Moreover, Sumiyoshi et al. showed that
the deletion of FcγRs preserves the endothelial
function and attenuates the oxidative stress induced by
hypercholesterolemia; moreover, FcγR-/-apoE-/- mice
had significantly smaller atherosclerotic lesions, a lower
content of macrophages and T cells, a reduced
expression of MCP1, ICAM1, and attenuated NF-κB
activation as opposed to control apoE-/- mice [116].
Toll-Like Receptors
TLRs are key pattern recognition receptors that
recognize pathogen-associated microbial patterns or
danger-associated molecular patterns, playing an
essential role in early innate immune responses to
invading pathogens or endogenous danger signals.
TLRs are expressed by various immune cells
(macrophages, dendritic cells, etc.) and non-immune
cells (fibroblasts, epithelial cells, etc.), as described in
[117]. The importance of cell-specific targeting of TLR
pathway is highlighted by the findings showing that
TLR-mediated TRAF6 signaling in EC promoted
atherosclerosis, while in macrophages it was
atheroprotective by modulating the anti-inflammatory
response and efferocytosis [118].
To date, ten human and twelve murine TLRs have
been identified and they are located on the plasma
membrane, excepting TLR3, TLR7-9, which are
8 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
localized in the endosomal compartment [119]. After
ligand binding, TLRs homo- or hetero-dimerize, recruit
the adaptor protein myeloid differentiation factor 88
(MyD88) and initiate signaling cascades leading to the
activation of transcription factors, such as NF-κB,
thereby influencing inflammatory responses [120].
MyD88 mediates the downstream signaling of TLRs,
excepting TLR3 [121].
Given the link between innate system responses,
inflammation and atherogenesis, and the fact that both
murine and human atherosclerotic plaques display an
enhanced expression of TLRs [117], it was
hypothesized that targeting TLRs or MyD88 could
produce beneficial effects against atherosclerosis.
First, MyD88-/-apoE-/- mice were generated and they
displayed an important reduction in development of the
atherosclerotic lesions associated with the decreased
macrophage recruitment into the artery wall [122, 123].
Surprisingly, a recent study failed to show detrimental
role of MyD88; instead it reported that the maturation of
dendritic cells mediated by MyD88 could provide
atheroprotection. Thus, Subramanian et al.
transplanted LDLR-/- mice with bone marrow from mice
in which MyD88 was deleted in CD11c+ dendritic cells
and observed less regulatory T cells in the
atherosclerotic lesions and an increase in the
atherosclerotic lesion size [124]. The proposed
atheroprotective mechanism of MyD88-mediated
dendritic cells activation occurs by promoting regulatory
T cells generation which in turn attenuates the
recruitment of monocytes by decreasing MCP-1
synthesis [124].
Considering the broad role of MyD88 in TLRs
signaling, targeting individual TLRs could represent a
more attractive therapeutic approach leading to more
specific outcomes. Important findings regarding the role
of individual TLRs in atherosclerosis are described
TLR2-/-apoE-/- mice as well as TLR2-/-LDLR-/- mice
displayed an important decrease of the number of
atherosclerotic lesions within aortas, demonstrating the
involvement of TLR2 activation in atherogenesis [125,
126]. Although complete deficiency of TLR2 in LDLR-/-
mice led to a reduction in atherogenesis, the loss of
TLR2 expression in bone marrow-derived cells did not
have an impact on atherosclerosis, suggesting that
cell-type specific TLR2 activation differentially
contributes to lesion development [125]. TLR2 in early
atherosclerotic lesions was found to be predominantly
of endothelial origin and its increased expression in
lesion-prone areas of the mouse aorta plays a role in
the progression of atherogenesis [127]. Recently,
Wang and coworkers [128] showed that knockout of
TLR2 gene or blocking TLR2 with neutralizing
antibodies reduces and stabilizes advanced
atherosclerotic plaques in apoE-/- mice by diminishing
the level of pro-inflammatory cytokines and inactivating
the NF-κB and STAT3. In addition, they noticed that
TLR2 blockade decreases CHOP expression and
inhibits the macrophage apoptosis induced by the
endoplasmic reticulum stress, and thus, inducing the
resolution of necrotic cores in advanced atheroma
plaques [128].
Unlike TLR2, TLR3 is MyD88-independent and
signals via Toll/IL-1R domain-containing adaptor
inducing IFN-β (TRIF), as recently revealed [129].
LDLR-/- mice with mutations affecting TRIF functionality
were protected from atherosclerosis [130]. Cole et al.
noted a significant increase in TLR3 expression and
activity in atheroma-derived SMC and they showed that
TLR3 deficiency accelerated early atherosclerosis in
apoE-/- mice, suggesting a protective role for TLR3 in
blood vessels [131]. Recently, Ishibashi et al. revealed
an important role for TLR3 in the extracellular matrix
degradation, in part by modulating the activities of
MMP-2 and MMP-9 in macrophages [132]. TLR3
stimulation induced endothelial dysfunction and
increased the atherosclerotic plaque development in
apoE-/- mice, suggesting a pro-atherogenic role of TLR3
[133]. In another study, Lundberg et al. reported that
hematopoietic deletion of the TLR-signaling pathways
involving TLR3, TRIF and TRIF-related adaptor
molecule reduces vascular inflammation and inhibits
the development of atherosclerosis [134].
TLR4 plays an important role in lipopolysaccharide
binding and can induce various inflammatory genes by
NF-κB activation [135]. Recently, it has been revealed
that oxidized LDL-induced expression of inflammatory
cytokines is decreased in SMC derived from TLR4-/-
mice [136]. Michelson and coworkers observed that
apoE-/- mice that also lacked TLR4 or MyD88 had
reduced aortic atherosclerosis [123]. In addition, they
noticed a reduction in the circulating levels of pro-
inflammatory cytokines as well as in the number of
macrophages in atherosclerotic plaques. Ding et al.
revealed that TLR4 deficiency decreased
atherosclerosis in LDLR-/- mice [137]. In contrast, an
atheroprotective role for TLR4 was suggested by the
increased susceptibility to atherosclerosis of TLR4-/-
apoE-/- mice after oral infection with the common
pathogen Porphyromonas gingivalis [138].
In contrast to the detrimental role of other TLRs, an
atheroprotective role was attributed to TLR7 and TLR9.
Salagianni et al. demonstrated that the functional
inactivation of TLR7 in apoE-/- mice accelerated the
development of atherosclerotic lesions interfering with
macrophage pro-inflammatory responses to TLR2 and
TLR4 ligands, and preventing the expansion of Ly6Chi
inflammatory monocytes and the accumulation of
inflammatory M1 macrophages within developing
atherosclerotic lesions [139]. TLR9-/-apoE-/- mice
displayed aggravated atherosclerotic lesions and the
CD4+ T cells were reported as possible mediators of
the atheroprotective effect; in addition, treatment with a
TLR9 agonist decreased the atherosclerotic lesions
TNF Receptors
The physiological as well as pathological effects of
TNF are mediated by two subtype receptors, TNFR1
and TNFR2 (also known as p55 and p75), which are
co-expressed on most cell types [141]. Experimental
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 9
data using p55 or p75 deficient mice revealed that p55
receptor mediates the majority of TNF inflammatory
responses [142]. Thus, consistent evidence indicated a
pro-atherogenic role for p55 TNFR [143, 144]. In order
to investigate the cell type specific role of p55 in
atherogenesis, Xantoulea and coworkers transplanted
LDLR-/- mice with bone marrow derived cells from p55
TNFR-/- mice, reporting that these mice presented a
reduced atherosclerotic plaque area and smaller
macrophage foam cells, as compared with the control
mice [145]. Additionally, deficiency of p55 TNFR in
bone-marrow derived cells was associated with a
important reduction in MCP1 systemic levels,
suggesting an important mechanism by which p55 may
contribute to atherogenesis. Therefore, selective
inactivation of p55 TNFR seems to be a promising
approach for atherosclerosis treatment.
Prostanoid Receptors
Thromboxane A2 (TxA2) and prostaglandin I2
(PGI2) belong to prostanoid group that plays a key role
in modulating the inflammatory responses and exert
their effects by activating G-protein coupled rhodopsin-
like receptors. The prostanoid receptor family is
involved in various biological processes [146]. TxA2
promotes platelet aggregation and acts as a smooth
muscle constrictor, while PGI2 inhibits aggregation of
platelets and relaxes vascular smooth muscles by
stimulating adenylate cyclase [147]. Besides their
expression on platelets, TxA2 and PGI2 receptors are
present on a variety of cell types, such as immune, EC
and SMC [148]. From the two thromboxane receptors
(TP) splice variants, only the TPα isoform is expressed
in mice. Thomas et al. generated TPα-/- mice, which
displayed a mild bleeding tendency and resistance to
platelet aggregation by TP agonists [149]. Since TxA2
and PGI2 prostacyclin biosynthesis was increased in
patients with atherosclerosis [150], the effect of TxA2
or PGI receptor blockade on atherogenesis was
assessed. Cayatte et al. [151] showed that the
inhibition of platelet TxA2 synthesis with aspirin had no
significant effect on atherogenesis, while inhibition of
the TxA2 receptor by S18886 antagonist caused a
reduction of advanced atherosclerotic plaques in apoE-
/- mice; the authors suggested that the mechanism by
which TxA2 receptor blockade with S18886 exerted
beneficial effects against atherosclerosis could involve
a decreased expression of the endothelial adhesion
molecules. Kobayashi and coworkers [152] noticed that
the suppression of atherosclerosis by TxA2 receptor
deficiency was much more robust than that found after
treatment with the S18886 antagonist. Despite similar
levels of serum cholesterol and triglyceride, they found
a significant reduction of atherosclerosis in TP-/-apoE-/-
mice and a marked acceleration of atherosclerosis in
IP-/-apoE-/- mice compared with control apoE-/- mice.
These data suggest that TxA2 enhances and PGI
suppresses the atherogenesis by the modulation of the
leukocyte-endothelial cell interaction and platelet
activation. Moreover, when translating these
discoveries into clinics, patients with coronary artery
disease treated with aspirin and receiving orally
S18886 displayed significantly improved endothelium-
dependent vasodi-lation in the peripheral arteries; the
authors suggested that S18886 treatment may partly
counterbalance overproduction of endothelial
vasoconstricting factors, and did not exclude a partial
restoration of endothelial relaxing factors production as
a mechanism to explain the beneficial effect of TxA2
receptor blockade on EC function [153].
Glucagon-Like Peptide-1 Receptors
Glucagon-like peptide-1 (GLP-1) is a hormone
released post-prandially from the intestine. By binding
to its receptor (GLP-1R) in the endocrine pancreas,
GLP-1 stimulates insulin secretion from β-cells and
inhibits glucagon secretion from α-cells in a glucose-
dependent manner and is rapidly degraded in the
circulation by the dipeptidylpeptidase-4 (DPP-4),
reviewed in [154]. GLP-1R is a G protein-coupled
receptor belonging to the same family as the gastric
inhibitory polypeptide and the glucagon receptors [155].
The GLP-1Rs are expressed on many cell types,
including macrophages, EC and SMC. GLP-1R
agonists and DPP-4 inhibitors were sought in a search
for type 2 diabetes treatment and as a way of delaying
hyperglycemia/hyperinsulinemia-associated atherogenic
processes. GLP-1R agonists (exenatide, exendin-4)
inhibited macrophage accumulation in the arterial wall
by inhibiting their inflammatory response and hence
attenuated atherosclerosis [156].
It was shown that GLP-1 induces macrophage
differentiation into the anti-inflammatory M2 phenotype
via STAT3 activation, which can induce the anti-
atherosclerotic effects of GLP-1 [157]. GLP-1R
activation with the agonist liraglutide protected against
endothelial dysfunction and vascular adhesion through
reduced expression of plasminogen activator inhibitor
type-1, vascular cell adhesion molecule and
intercellular adhesion molecule-1 and increased nitric
oxide synthase activity in human vascular EC in vitro
and in apoE-/- mice [158]. These results were further
supported by other studies of liraglutide administration
in atherosclerotic apoE-/- mice, where benefits were
noticed depending upon the stage of atherosclerosis
when the treatment was initiated. Hence, liraglutide
inhibited the progression of the lesion in early stages in
a partially GLP-1R-dependent manner, but had no
effect on later events [159]. The DPP-4 inhibitor
vildagliptin provides a significant anti-atherosclerotic
effect in both non-diabetic and diabetic mice apoE-/-
mice, mainly via increased GLP-1. However, the effect
was only partially attenuated by antagonizing the
receptor, indicating that other receptor independent
events may occur when inhibiting DPP-4. Long-acting
GLP-1R agonist taspoglutide was reported to lower
triglyceride level in apoE-/- mice, by modulating the
expression of hepatic genes controlling fatty acid
uptake and oxidation, without affecting plaque area or
lipid content in aorta [160]. A series of GLP-1 split
products reduced inflammation and increased plaque
stability in the apoE-/- mouse model, despite the fact
that these peptides did not activate the receptor [161].
10 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
In conclusion, these pharmacological studies point
towards an atheroprotective effect of GLP-1R, when
specifically targeted.
Prolactin Receptors
Prolactin is a pituitary-derived hormone known for
the main function of ensuring lactation. The 23 kDa
polypeptide hormone binds its receptor which belongs
to the cytokine receptor superfamily, reviewed in [162].
Today, besides its endocrine function, prolactin
appears to play a new autocrine/paracrine role since
both the hormone and its receptor are also produced in
extra-pituitary sites, such as: lymphoid cells [163], EC
[164], SMC [165]. Multiple isoforms of the prolactin
receptor with different lengths of their intracellular
domains have been described as result of alternative
splicing in humans, mice and rats. They bear different
number of tyrosine residues, leading to differential
phosphorylation status. Certain isoforms seem to
exhibit tissue-specific expression, like the short form of
the receptor in the liver; the long 558 amino acid form
of the receptor is involved in various signaling
cascades, such as Jak-STAT, MAPK, and PI3K,
leading to differentiation, proliferation, survival, and
secretion [162].
It was shown through knockout studies on the
prolactin receptor in mice that the prolactin signaling
pathway affected insulin production in pancreas [166]
and accumulation of fat in adipose tissue [167]. It has
been suggested that effects of prolactin on metabolism
and inflammation could influence atherogenesis and
cardiovascular disease. Although elevated systemic
prolactin levels seem not to predict coronary artery
disease in the general population, an enhanced
expression of prolactin receptor has been reported in
advanced atherosclerotic lesions [168, 169]. These
observations could indicate a role of prolactin within
atherosclerotic plaques. To test if inhibition of prolactin
may have anti-atherogenic potential, atherosclerosis-
prone LDLR-/- mice were transplanted with bone
marrow from transgenic mice expressing the pure
prolactin receptor antagonist, Del1-9-G129R-hPRL
[170]. The data showed no significant differences in the
size of the atherosclerotic lesions, in macrophage
accumulation and in collagen amount found in the
lesions after 12 weeks of Western diet feeding, as
compared with the control group. However, recipient
mice expressing the antagonist Del1-9-G129R-hPRL
showed a reduction in plasma cholesterol that can be a
consequence of the significant decreased level of the
cholesterol esters associated with VLDL/LDL; by
contrast, there were increased number of circulating
neutrophils, lymphocytes and monocytes. Probably,
this increased number of leukocytes shielded the
lowering effect of prolactin receptor antagonist on
cholesterol level and thus, no total effect was seen on
the atherosclerotic lesions [170].
More studies are needed to define the possible role
of prolactin and its receptor in atherosclerotic plaque
Leptin Receptors
Leptin (product of ob gene) is an adipocyte-derived
hormone that plays an important role not only in body
weight regulation [171], but also in other metabolic and
immune processes [172]. Leptin receptors (products of
db gene) are members of the class I cytokine receptor
family that utilize Jak-STAT signal transduction
pathway [173, 174]. Six isoforms bearing the same
extracellular domain were described: five membrane
bound (one long, four short) and one plasma soluble
variant. The long biologically active form is expressed
in many sites as: hypothalamus, immune cells, SMC,
hematopoietic cells [175], coronary EC [176].
The role of leptin and leptin receptor signaling in
vascular disease remains controversial, since in
studies using genetically modified animals, leptin/leptin
receptor deficiency either accelerated or prevented
atherosclerosis, probably depending on the genetic
background and/or other factors. It was reported that
leptin treatment of apoE-/- mice resulted in enhanced
atherosclerosis and more rapid occlusive thrombosis
after vascular injury [177], and promoted osteogenic
differentiation and vascular calcification [178], and thus,
leptin was associated with an increased cardiovascular
risk. However, in the diabetogenic Akita (Ins+) mice on
an apoE-/- background, leptin administration proved
beneficial in reducing atherosclerosis [179]. Ablation of
the long-form leptin receptor in apoE-/- mice fed a
regular chow diet led by the age of 20 weeks to the
development of the typical features of type 2 diabetes
and to accelerated atherosclerosis, as compared with
the apoE-/- littermates [180]. Additionally, administration
of Western diet further enhanced atherosclerosis, but
the fenofibrate treatment significantly reduced it in male
and, even more, in female mice [180]. A direct link
between leptin and apoE was recently provided by the
finding that leptin requires the presence of apoE,
expressed, secreted and bound to the cell surface in
order to activate leptin receptor signaling and to
promote SMC proliferation and neointima formation
On LDLR-/- background, leptin deficiency was
reported to either enhance [182] or reduce the
atherosclerotic lesions [183]. The macrophage leptin
receptor transplantation did not improve aortic root
atherosclerotic lesion formation in a double knockout
Leprdb/dbLDLR-/- [184]. Certain single nucleotide poly-
morphisms of the leptin receptor have been described
to affect incidence of cardiovascular disease, reviewed
in [185]. Since some mutations occur in the domain not
involving direct leptin binding, it appears that the
downstream signaling would be responsible for such
effects. Thus, mice with selective deficiency of leptin
receptor-STAT3 signaling (Leprs/sapoE-/-) were signi-
ficantly protected from atherosclerosis compared to the
mice completely deficient in leptin receptor signaling
(Leprdb/dbapoE-/-), as recently revealed [186].
Nicotinic Acid Receptors
Nicotinic acid (niacin) has been known for decades
to lower plasma cholesterol in humans [187]. Niacin
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 11
effects on lipid profile include lowering atherogenic
LDL- and VLDL-, while increasing HDL-cholesterol
[188], this broad lipid modulation making it an ideal
anti-atherosclerosis therapeutic agent. However, niacin
is still underused in atherosclerosis mainly due to the
otherwise harmless flushing side effect, which leads to
loose patient adherence to therapy. However, the
recent involvement of niacin in anti-inflammatory
signaling [189] renewed the interest toward this
To date, it appears that the pleiotropic actions of
nicotinic acid are partly mediated through its seven-
transmembrane high affinity receptor GPR109A [190].
It was found that GPR109A also functions as the
receptor for the endogenous ketone body 3-hydroxy-
butyrate, hence its alternative naming as hydroxy-
carboxylic acid receptor 2 (HCA2). GPR109A is
expressed in adipocytes, neutrophils, macrophages,
keratinocytes and Langerhans cells, as reviewed in
[191]. The mechanism of action of niacin on plasma
lipid level seemed to occur at least in part via
GPR109A activation, a Gi/Go protein-coupled receptor,
which reduces the levels of intracellular cAMP,
inhibiting lipolysis in adipocytes and leading to less free
fatty acids (FFA) [192] available for liver syntheses
[193]. Yet, this “FFA hypothesis” was recently
challenged in mice and humans. In mice lacking
GPR109A, upon treatment with niacin, the anti-lipolytic
effect was still present, while there was no lowering of
plasma lipids. Same uncoupling was reported in clinical
trials in humans upon administration of two GPR109A
full agonists: MK-1903 and SCH900271 [194]. New
data confirm that the anti-atherosclerotic effect of niacin
is mediated by GPR109A, but rather through immune
cells independent of its anti-dyslipidemic effect. Thus,
in GPR109A-/-LDLR-/- mice exposed to Western type
diet in the presence of niacin, no reduction of the
lesions was seen as compared with LDLR-/- control
animals. When bone marrow from wild type or
GPR109A-/- mice was transplanted into irradiated
LDLR-/- donors, no beneficial effect of niacin was
reported in the GPR109A-/- chimeric mice fed with high
fat diet. This clearly identified GPR109A on lymphoid
cells as responsible for the anti-atherosclerotic effect of
niacin. Moreover, macrophage GPR109A was found to
be responsible for cholesterol efflux via nicotinic acid-
induced expression of ABCG1 transporter and impaired
the recruitment of macrophages to atherosclerotic
plaques via MCP-1 inhibition, underlining the import-
ance of anti-inflammatory effects of the receptor [195].
Other anti-atherosclerotic effects of nicotinic acid
are a consequence of its action on the receptor on the
vascular endothelium. Very recently, GPR109A
expression has been identified in human microvascular
EC, where it mediated the niacin-induced angiogenesis
under lipotoxic conditions, as demonstrated by the use
of the acifran MK-1903 agonist and GPR109A siRNA
[196]. Significant for its therapeutic use, the flushing
effect of nicotinic acid was found to be mediated via the
ERK 1/2 MAP kinase cascade induced by β-arrestins
involved in receptor recycling [197]. In turn, activation
of MAP kinase causes release of prostaglandin D2
from Langerhans cells in the skin. Thus, while the
cAMP-mediated effects of nicotinic acid occur via
clathrin-dependent internalization of the receptor, the
flushing effects are the consequences of an alternative
downstream signaling. This can be viewed as a
therapeutic opportunity for designing preferential
(“biased”) ligands both for nicotinic acid as well as for
other drugs which signal via multiple pathways.
Cannabinoid Receptors
Derivatives of cannabinoids (CBs) have anti-
inflammatory potential [198-200], and their biological
effects are transduced by two distinct G-protein
coupled receptors, the peripheral CB1R and the central
CB2R [201, 202]. CB1Rs are highly expressed in brain
and at lower levels in immune cells and peripheral
tissues [203]. CB2Rs are mainly expressed in immune
cells such as T lymphocytes and macrophages within
murine and human atheromatous lesions [204, 205].
GPR55, an orphan G-protein-coupled receptor, with
low sequence homology compared to that of CB1R and
CB2R, has been reported as a third putative CBR, but
further studies are needed in order to demonstrate that
this is a true cannabinoid receptor [206, 207].
Using apoE-/- mice, Steffens et al. reported that oral
administration of low doses of delta-9-tetrahydro-
cannabinol (THC) resulted in a significant inhibition of
atherosclerosis progression [200]. THC-treated apoE-/-
mice displayed a decreased macrophage content
compared with untreated mice; moreover, the results of
in vitro studies showed that macrophage chemotaxis
was reduced by THC treatment [200]. The inhibitory
action of THC was reversed in the presence of CB2R
antagonist SR144528, but not by SR141716, a CB1R
antagonist [208]. CB1R activation promotes cholesterol
accumulation, release of inflammatory mediators and
vascular SMC proliferation, representing a risk factor in
atherosclerosis [209, 210]. Accordingly, the selective
CB1R antagonist rimonabant reduced plasma levels of
the pro-inflammatory cytokines and attenuated the
atherosclerosis in LDLR-/- mice [211]. Conversely,
CB2R deficiency in LDLR-/- mice did not affect the area
of the atherosclerotic lesion, but increased macrophage
and SMC accumulation and altered extracellular matrix
composition, suggesting a role for CB2R in modulating
cellular composition and stability of atherosclerotic
plaques [212]. Lack of CB2R in hematopoietic cells
aggravated early atherosclerosis in LDLR-/- mice,
indicating a protective effect for CB2R in atherogenesis
[213]. Hoyer and coworkers observed increased ROS
level and leukocyte infiltration in atherosclerotic
plaques in CB2R-/-apoE-/- mice compared with apoE-/-
mice, reporting that the atheroprotective effects of
CB2R are mediated mainly by circulating and vascular
cells [214]. Accordingly, the same authors noticed that
apoE-/- mice treated with the selective CB2 agonist
JWH133 during a high cholesterol diet displayed a
reduction of the atherosclerotic lesions, improved
endothelial function as well as lower levels of ROS
[214]. It is noteworthy that numerous studies indicated
the anti-inflammatory and protective role of CB2R in
12 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
atherosclerosis as well as other cardiovascular
diseases [66, 215, 216].
Plasminogen Receptors
Plasminogen receptors (Plg-Rs) represent a
heterogeneous group of proteins able to bind
plasminogen (Plg) as well as plasmin. The members of
this group are integral membrane proteins, but also
proteins with cytosolic or nuclear localization. Plg-Rs
are involved in a variety of biological processes
comprising inflammation, angiogenesis, wound healing,
cell differentiation [217-219]. The cell membrane
receptors for plasminogen, out of which the most
studied is Plg-RKT, are localized on cells directly
involved in the atherosclerotic process. Besides
plasmin production, plasminogen activation induces
vascular SMC apoptosis in the atherosclerotic lesion
[220]. Furthermore, plasmin stimulates cell migration
and modulates the activity of MMPs and several growth
factors, such as TGF-β [221, 222]. Xiao et al. reported
aggravation of intimal atherosclerotic lesions in
Plg-/-apoE-/- mice, while no significant atherosclerosis
was noticed in Plg-/- mice [223]. Plg-RKT is a
transmembrane protein with a C-terminal lysine
exposed on the cellular surface, with role in plasmin
binding [224]. Plg-RKT is expressed on monocytes and
macrophages [217, 218], modulating the macrophage
recruitment in the inflammatory response [225]. Despite
the evidence of Plg-Rs involvement in a variety of
processes that take place during atherogenesis
(inflammation, monocytes recruitment, ROS
production, cytokine induction, etc.), on our knowledge,
Plg-Rs knockout mice on background of atherosclerotic
model were not obtained yet, and thus, the role of Plg-
Rs as putative target in atherosclerosis remains to be
Serotonin receptors
Serotonin (5-hydroxytryptamine, 5-HT) receptors
comprise a family of seven classes with a total of 14
subtypes, mostly of the G protein-coupled receptors
(GPCRs), with the exception of 5-HT3, which belongs
to the ligand-gated ion channels (LGICs). GPCRs
employ cAMP as second messenger, only 5-HT2 is
IP3/DAG based. For a comprehensive structural and
physiological description of 5-HT receptors, the reader
is directed to the reviews of Hoyer [226] and that, more
recent, of Nichols [227]. 5-HT receptors mediate both
excitatory and inhibitory neurotransmission in the
central and peripheral nervous systems upon binding of
their endogenous ligand serotonin. Not only is
serotonin involved in cognition-related processes [228],
but it also mediates vasoconstriction in the periphery,
and is linked to hypertension and inflammatory
processes which promote atherosclerosis [229]. Thus,
serotonin from activated platelet stores is released into
plasma, promoting platelet aggregation in an autocrine
loop via 5-HT2A receptor. Serotonin acts on the 5-
HT2A receptor on vascular muscle cells and promotes
vasoconstriction and proliferation [230]. However, on
endothelial cells, 5-HT induces an increased production
of the vasodilator NO, via the 5-HT1B receptor [231].
Therefore, it seems that the vascular tone is poised by
the VSMC 5-HT2A and the endothelial 5-HT1B
receptor subtypes [232]. Plasma serotonin level was
suggested as an indicator of the endothelial function in
patients with mild atherosclerosis [233]. Early studies
demonstrated that synthetic antagonists of serotonin
receptors, such as MCI-9042, protected from experimental
thrombosis in mice [234, 235]. It was shown that
serotonin upregulated in a dose-dependent manner
acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-
1) in monocytes/macrophages, via the 5-HT2A
receptor/G protein/c-Src/PKC/MAPK pathway. Sero-
tonin-induced upregulation of ACAT1, which is
responsible for the formation of macrophage-derived
foam cells in atherosclerotic lesions, was abrogated by
the use of sarpogrelate, a selective 5-HT2A receptor
antagonist [236]. Sarpogrelate displays pleiotropic
effects. Thus, it is used as an anti-platelet drug to
improve peripheral arterial disease [237].
Administration of sarpogrelate simultaneously with
pioglitazone treatment in diabetic KK-A male mice
improved the therapeutic outcome by preventing body
weight gain induced by pioglitazone itself [238]. In rats
with streptozotocin-induced diabetes, sarpogrelate
increased the expression of the membrane GLUT-1
and GLUT-4 glucose transporters, and promoted the
release of pancreatic insulin [239]. Sarpogrelate
induced insulin-sensitization in patients with peripheral
arterial disease by changes in adiponectin [240]. In
contrast, systemic sarpogrelate administration
repressed hyperglycemia in obese A(y) mice, but it did
not raise plasma adiponectin [241]. Sarpogrelate also
reduced blood viscosity, the levels of cholesterol and
triglycerides and oxidative stress in high-cholesterol fed
rabbits [242]. Sarpogrelate acted synergistically with
multipotent adipose-derived stromal cells in restoring
angiogenesis and the capacity to modulate
inflammation in ischemic hindlimb of aged mice, by
preventing hypoxia/reoxygenation-induced apoptosis of
the stromal cells via a mTOR/STAT3-dependent
pathway in vitro [243]. From these and other data from
the literature, it appears that sarpogrelate may be a
useful therapeutic agent from atherosclerosis and
Recent evidence indicates that plaque angiogenesis
contributes to the development of an unstable or
rupture-prone atheroma plaque. Consequently, the
identification of factors that inhibit the aberrant
angiogenesis is essential in the development of novel
strategies used to combat atherosclerosis and its
complications. Among the most important receptors
modulating angiogenesis, we focused below on the
involvement of angiopoietin receptors, FGFR, PDGFR,
VEGFR and tyrosine receptor kinase in atherogenesis.
Angiopoietin Receptors
The orphan receptor Tie1 and the endothelial-
specific receptor Tie2 are tyrosine kinase receptors
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 13
with a key role in EC proliferation and survival during
angiogenesis [244]. Despite their considerable
similarity, studies using transgenic mice have provided
major insights into the distinct expression and functions
of Tie1 and Tie2. Numerous lines of evidence indicated
that Tie1 is almost exclusively expressed in EC and
hematopoietic lineage, while Tie2 is expressed in EC
and their precursors in both embryonic and adult
vasculature in many tissues [245, 246]. Interestingly, it
has been reported that Tie2 is necessary for Tie1
activation and the interaction of Tie1 with Tie2
modulates Tie2 activation [247, 248]. Elegant studies of
Seegar et al. revealed the direct interaction between
Tie1 and Tie2 on the endothelial cell surface,
suggesting that Tie1 plays an essential role in
angiopoietin-induced Tie2 signaling [249]. An increased
expression of Tie1 was detected in pro-atherogenic
shear stress areas, specifically at the bifurcations of
aortic branches and microvasculature, indicating the
involvement of Tie1 in development and progression of
atherosclerosis [250-252].
Two groups independently showed that Tie1
ablation in transgenic mice resulted in embryonic
lethality in early stages, suggesting that Tie1 is
required for the EC integrity and survival during
ontogenesis [253, 254]. The hypothesis of Tie1
involvement in endothelial response to the shear stress
was proven using Tie1+/-apoE-/- mice that displayed a
considerable reduction of atherosclerosis [251]. In
addition, Woo et al. used an inducible and conditional
Tie1 mouse model and revealed that endothelial-
specific Tie1 deficiency reduces atherogenesis
depending on the dose. Tie1 attenuation was
accompanied by an increase in Tie2 phosphorylation,
eNOS and IκBα expression, and a reduction of ICAM
levels [251]. The results of in vitro studies using human
EC showed that small interfering RNA knockdown of
Tie1 reduced the expression of many pro-inflammatory
genes [255].
Disrupting Tie2 function in mice resulted in embryonic
lethality, suggesting a critical role of Tie2 in later stages of
embryonic vascular development and in adult vasculature
[256]. Using a soluble Tie2 extracellular domain
designated to block the activation of Tie2, Peters et al.
showed that inhibition of Tie2 signaling pathway has
therapeutic effects in rodent tumor models [257]. In 2009,
Hauer et al. created a vaccination strategy of LDLR-/- mice
against Tie2, which attenuated and stabilized the
atherosclerotic lesions [258].
Fibroblast Growth Factors Receptors
Fibroblast growth factors are found on a wide range
of cell types and play a key role in various processes:
early embryonic stages, neovascularization, wound
healing, inflammation and tumor growth [259]. To date,
more than twenty FGFRs were identified, many of them
being expressed in the atherosclerotic plaques; FGFR1
and FGFR2 are considered the major activated FGFR
types that contribute to lesion development [260, 261].
The results of previous studies in apoE-/- mice
expressing constitutively active FGFR2 in endothelium,
as well as studies using endothelium-targeted
transgenic mice clearly demonstrated that, upon
activation, endothelial FGFR2 has anti-proliferative and
pro-apoptotic effects, aggravating the extent of
atherosclerosis [262, 263]. Several compounds found
to inhibit FGFRs were used to answer the question on
the effect of systemic FGFR blockade in mouse models
of atherosclerosis. FGFR tyrosine kinase inhibitor
SU5402 attenuated the progression of atherosclerosis
in apoE-/- mice on Western diet [264]. However, since
SU5402 compound was also found to be a potent
inhibitor of VEGFR activation, it is difficult to
unequivocally ascribe its effects to FGFR or VEGFR
inhibition [265]. A selective FGFR2 antagonist
SSR128129E slightly decreased early atherosclerotic
lesions in apoE-/- mice, but the compound was much
more active against advanced lesions [265]. Besides
the reduction of atherosclerosis progression, oral
treatment of apoE-/- mice with this synthetic FGFR
antagonist decreased neointimal proliferation after a
vein graft procedure, suggesting that selective FGFR
inhibition may have beneficial effects in atherosclerosis
and other vascular diseases [266].
Platelet-Derived Growth Factor Receptors
PDGFs are pleiotropic growth factors, synthesized
and secreted by activated platelets and other activated
or injured cells [267]. The five identified isoforms of
PDGF (AA, AB, BB, CC, DD) act via two tyrosine
kinase receptors, PDGFR-α and PDGFR-β [268].
PDGFR-α and PDGFR-β are highly expressed in
atherosclerotic vascular SMC, but the mechanism
leading to increased PDGFR expression in
atherosclerosis is unclear. It has not been possible to
analyze the effect of targeted deletion of PDGF
receptors in mice because of embryonic lethality;
however endothelial and hematopoietic cell disruption
of PDGFR-β generated viable mice and confirmed its
role in regulating SMC migration in vascular
pathologies [269, 270]. To study whether blockade of
PDGF or its receptors can prevent atherosclerotic
lesion formation in mice, Kozaki et al. used
hematopoietic chimeras with PDGF-β ablation in their
circulating cells or apoE-/- mice treated with CT52923, a
PDGFR antagonist [271]. Despite a delay in SMC
accumulation, neither method of blockade was able to
prevent formation of advanced fibrous caps. Moreover,
studies using hematopoietic PDGFB-/-apoE-/- chimeras
showed an increased accumulation of monocytes and
elevated numbers of activated T lymphocytes [272].
Sano et al. showed that the size of the atherosclerotic
lesions as well as the number of intimal SMCs were
reduced in apoE-/- mice injected with anti-PDGFRβ
antibodies, but not in mice injected with anti-PDGFRα
antibodies [273]. These findings suggest a possible
role of PDGFB in modulation of T-cell activation and
atherogenesis in mice. Treatment with imatinib (STI-
571), a PDGF antagonist, reduced the total
atherosclerotic lesion area as well as the infiltration of
SMCs and macrophages in the plaques of diabetic
apoE-/- mice [274].
14 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
Vascular Endothelial Growth Factor Receptors
VEGFs are considered as pivotal regulators of
vasculogenesis and angiogenesis. Although VEGFs act
mostly on EC, VEGF receptors are also expressed on
hematopoietic stem cells, monocytes, osteoblasts and
neurons [275]. To date, five VEGF family members and
three VEGF receptors have been identified: VEGFA,
VEGFB and PLGF (placental growth factor) bind to
VEGFC and VEGFD bind to VEGFR3. VEGFR1 is a
positive regulator of monocyte/macrophage migration
as well as a regulator of VEGFR2 signaling, VEGFR2
is involved in normal and pathological vascular
endothelial cell biology, while VEGFR3 plays a role in
lymphatic endothelial cell development and function
[276]. Celletti et al. observed that treatment of
cholesterol fed apoB-/-apoE-/-mice with low doses of
VEGF increased macrophage and endothelial cell
content of plaques and significantly induced
atherosclerotic plaque progression; these effects were
not species-specific, since increased atherosclerosis
development and angiogenesis was also noticed in
cholesterol-fed rabbits [277]. Afterwards, the effects of
VEGF-A, -B, -C, and -D on atherogenesis were tested
in apoB48-/- LDLR-/- mice, in which lipoprotein profile
more closely resembles that of humans [278].
However, the adenovirus-mediated gene transfers of
VEGF-A, -B, -C, and -D had no significant effect on
atherosclerosis development in hypercholesterolemic
apoB48-/-LDLR-/- mice.
Tyrosine Receptor Kinase
These receptors were discovered as important
players in the development of nervous system [279].
Three types of Trk receptors (TrkA, TrkB, TrkC) were
identified, each of them having different binding affinity
to certain types of neurotrophins. After ligand binding
they initiate various signaling pathways and finally
generate different biological responses. TrkB receptors
are expressed by hippocampal neurons and bind with
high affinity to the brain-derived neurotrophic factor
(BDNF), a growth factor with important role in the
function of neurons in the central nervous system.
Kraemer and coworkers, working on haplodeficient
TrkB+/-apoE-/- mice, revealed that diminished level of
TrkB receptor induced smaller atherosclerotic lesion
area, concomitant with a reduction in SMC accumulat-
ion; moreover, bone marrow transplantation from the
haplodeficient mice into apoE deficient mice (with
normal TrkB expression) did not affect atherogenesis
[280]. These data demonstrate the contribution of TrkB
receptors expressed by SMC to atheroma development
and the fact that they are not implicated in monocyte
migration and recruitment in the atherosclerotic plaque.
Ligand-activated TrkB receptors trigger intercellular
cascades and regulate neuronal development and
apoptosis, thus being associated with Alzheimer's
disease [281]. It is notable that, through TrkB
activation, BDNF promotes survival of EC and
angiogenesis [282]. Since BDNF-/- mice die after birth
[283], to investigate the effect of BDNF deficiency on
atherogenesis, a mouse model with a conditional
deletion of BDNF in EC on apoE-/- background was
generated; however similar atherosclerosis lesions
were developed, as compared with control apoE-/- mice
[284]. To date, the literature revealed that the
involvement of Trk receptors in atherosclerosis is
dependent on cell type distribution, emphasizing the
pro-atherogenic role of Trk receptors expressed by
The multifactorial origins of atherosclerosis as well
as the involvements of various contributors in the
progression of the atherosclerotic lesions indicate
additional targets to slow down or diminish the
atheromatous plaque. Some important anti- or pro-
atherogenic receptors involved in various phases of
atherogenesis are described below.
Nucleotide/ Nucleoside (Purinergic) Receptors
Extracellular nucleotides / nucleosides are released
from cells upon oxidative stress, inflammation, hypoxia,
ischemia, mechanical constraints, leading to the
activation of various responses as cell adhesion,
inflammation, altered glucose and lipid metabolism.
Nucleotides / nucleosides exert their action through
their plasma G-protein coupled (P1, P2Y) or ion-gated
(P2X) plasma membrane receptors. P1 and P2Y
receptors are known to be widely distributed in the
brain, heart, kidneys, and adipose tissue.
Adenosine receptors (AR or P1 receptors) belong to
a group of purinergic G-protein coupled receptors,
having adenosine as endogenous ligand, either
activating (A2a and A2b) or inhibiting (A1 and A3)
adenylyl cyclase. Adenosine is essential for the
following actions: regulation of the oxygen supply on
demand, tissue repair, anti-inflammatory response and
pro-angiogenic activity, which may be beneficial or not,
depending upon the physiological status, as reviewed
in [285]. Adenosine receptors are expressed by EC,
vascular SMC, macrophages [286].
A1AR. In the heart, stimulation of the adenosine A1
receptor decreases heart rate, a mechanism which
may have negative effects in pathological conditions
such as cardiac arrest, where a compensatory increase
in heart rate is needed. Ablation of A1AR in the A1AR-/-
apoE-/- mice led to reduced atheromatous plaques in
the aortic root, aortic arch and innominate arteries
compared to apoE-/- mice of the same age; proliferating
cell nuclear antigen (PCNA) expression and plasma
cytokine concentrations were also significantly lower in
double-knockout mice [287]. In addition, the same
authors revealed that the treatment with the A1AR
antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX)
also caused a reduction of the lesions in a concentration-
dependent manner. Together, these data point to a
pro-atherogenic action of A1AR, based on its pro-
inflammatory and mitogenic effects [287]. Recently,
using a A1R specific agonist/antagonist as well as
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 15
siRNA, Umapathy et al., showed that A1R to improve
vasa vasorum EC barrier integrity [288].
A2aAR. Most of the anti-inflammatory effects of
adenosine occur via A2aAR. Ablation of A2aAR in apoE-
deficient (A2aAR-/-apoE-/-) mice led to hypercholesterolemia
and enhanced the pro-inflammatory status of atheroscl-
erotic lesions; however, the double knockout A2aAR-/-
apoE-/- mice developed smaller lesions, as did chimeric
apoE-/- mice lacking A2aAR in bone marrow-derived
cells [289]. Despite that the homing ability of A2aAR
deficient monocytes did not change, the lesions of
these mice displayed a lower density of foam cells, due
to p38MAPK-dependent apoptosis [289]. These data
suggested that inactivation of A2aAR, especially in bone
marrow derived cells, inhibits the formation of
atherosclerotic lesions, making A2aAR inactivation a
useful approach for the treatment of atherosclerosis.
A2bAR. The lowest affinity adenosine receptor,
A2bAR is abundantly expressed in the vasculature,
macrophages, liver [286]. In the A2bAR-/-apoE-/- mice
fed with a Western diet an increase in plasma and liver
lipids levels and an augmentation of the atherosclerotic
plaque features (size of the lesions, lipid load) were
noticed; A2bAR ablation is consistent with a decrease in
cAMP, previously known to inhibit activation of SREBP-
1, therefore leading to an increased expression of this
transcription factor and of two of its downstream targets
involved in lipogenesis: acetyl CoA carboxylase and
fatty acid synthase [290]. Adenovirus- mediated hepatic
restoration of A2bAR in double knockout mice led to the
reduction of plasma lipids level and atherosclerotic
plaques [290]. The possibility of controlling A2bAR with
selective agonists makes this molecule an attractive
target to control the progression of atherosclerosis
even when no significant changes in the diet are
A3AR appears not to significantly influence
atherosclerosis, despite a proliferating effect on SMC
P2Y receptors belong to a family of purinergic G
protein-coupled receptors, which are stimulated by
nucleotides, such as ATP, UTP, ADP, UDP and UDP-
glucose. These receptors regulate the vascular tone,
SMC proliferation as well as inflammatory responses.
P2Y receptors are found in the blood vessels in EC,
SMC and leukocytes; in addition, P2Y receptors were
detected in the atherosclerotic lesions of apoE-/- mice,
where the expression of some of them was affected by
the progression of the disease [292].
P2Y1 receptor functions as a receptor for
extracellular ATP and ADP. In platelets, ADP binding to
the receptor causes the mobilization of intracellular
calcium ions via activation of phospholipase C, a
change in platelet shape, and probably in platelet
aggregation [293]. Whole body or organ-targeted
ablation of the P2Y1 receptor in apoE-/- mice showed
the involvement of the non-hematopoietic (especially
EC or SMC) P2Y1 receptors in enhancing atheroscle-
rosis [294]. Also, knocking out or antagonizing
endothelial P2Y1 receptors on an apoE-deficient
background significantly decreased TNF-induced
inflammation, due to a reduction of adhesion molecules
levels and subsequent lower leukocyte recruitment
P2Y2 receptor is responsive to both ATP and UTP
and is reported to be overexpressed on some cancer
cell types. It is involved in many cellular functions, such
as proliferation, apoptosis and inflammation. In
addition, its implication in the spreading and migration
of SMC was shown [296, 297], yet its level was not
affected by atherogenic events [292].
P2Y6 is a Gq-protein coupled receptor activated
solely by UDP, whose expression is specifically
increased in the plaques [292]. UDP binds to P2Y6 and
propagates inflammation in the vasculature by
induction of various chemokines expression, and thus
contributing to leukocyte recruitment and inflammatory
states. P2Y6
-/- mice have impaired response to UDP in
macrophages, EC, and vascular SMC [298]. In order to
delineate P2Y6 role in atherogenesis, the effects of
whole body ablation of the receptor were examined by
two independent studies on P2Y6
-/-LDLR-/- mice fed a
pro-atherogenic diet, one reporting a smaller size of the
lesions in the case of P2Y6 deficiency [299], while the
other reported no significant effect [300]. However,
both groups described similar results when bone
marrow transplantation experiments were performed:
whole-body irradiated LDLR-/- mice receiving bone
marrow-derived stem cells from P2Y6
-/- donors showed
a significant decrease in plaque formation compared
with the P2Y6
+/+ recipients. These results were
consistent with the pro-inflammatory pro-atherogenic
role of P2Y6.
P2Y12 is a ligand for ADP and is involved in platelet
aggregation, being a therapeutic target for clotting
disorders. Studies with P2Y12-/-apoE-/- mice showed
not only the role of platelet P2Y12 in atherogenesis via
leukocyte recruitment and infiltration, but also a
possible role of the receptor expressed on SMC [301].
P2Y13 was recently identified as being directly
involved in reverse cholesterol transport [192], and thus
it has become a candidate for novel therapeutic
P2X receptors, their role in the cardiovascular
system and their therapeutic potential has just been
reviewed elsewhere [302]. P2X receptors are ATP-
gated ion channels, assembled in homo- or hetero-
trimers through combinations of seven protein subunits
(P2X1-7), encoded by different genes. All P2X receptors
are permeable to small monovalent cations and some
display calcium or anion permeability. Several subtypes
(P2X2, P2X4, P2X7) become permeable even to larger
molecules (up to 900Da) upon prolonged ATP
exposure. P2X receptors are strongly expressed in the
nervous system, vascular system and hematopoietic
tissues. We overview below only the P2X receptors
whose link to cardiovascular pathology is most
P2X1 is expressed in smooth muscle, being
responsible for vasoconstriction in small arteries and
16 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
arterioles. Platelet P2X1 contributes to platelet
aggregation and thrombus formation, together with the
other two platelet purinergic receptors P2Y1 and P2Y12
[303]. Inhibiting these receptors in knockout studies or
with selective antagonists is sought as an effective
antithrombotic therapy to help atherosclerotic patients,
as reviewed in [304].
P2X4 is differentially expressed in the vasculature,
being highly expressed in veins compared to arteries.
Since P2X4 is implicated in calcium influx into EC,
blood vessel contractility and intima proliferation, its
selective localization could explain why vein grafts are
more susceptible to developing atherosclerosis [305].
Ablation of P2X4 in the heart depressed contractile
performance in models of cardiac failure, whereas
P2X4 overexpression proved to be cardioprotective
[306], and thus P2X4 may serve as a therapeutic target
in the treatment of heart disease.
P2X7 receptors represent a link between the
nervous, cardiovascular and immune systems through
their ability to regulate interleukin-1β processing and
release. P2X7 receptors were initially found in
hematopoietic cells to mediate the influx of Ca2+ and
Na+ ions. P2X7 activation promotes inflammatory
responses, as demonstrated by their attenuation in
P2X7-deficient mice. This mechanism may be
responsible for neuronal and endothelial cell apoptosis
[307]. Under inflammatory conditions, EC up-regulate
their production of P2X4 and P2X7. Activation of P2X7
led to simultaneous production of both pro- and anti-
inflammatory IL-1 receptor ligands (IL-1β, respectively
IL-1 receptor antagonist IL-1Ra), the ratio between
them acting as a switch in controlling the inflammatory
status of the arterial wall [308]. P2X7 are also
expressed and active in human adipocytes, where they
modulate the release of inflammatory cytokines (TNFα,
IL-6 and PAI-1), as demonstrated via inhibition by the
specific human P2X7 blocker, KN62 or by P2X7 gene
silencing. Adipocytes from patients with metabolic
syndrome present an upregulated P2X7 expression,
and thus the sustained inflammation in these patients,
which leads to an increased cardiovascular risk, could
be explained [309]. P2X4 and P2X7 are implicated in
ADP release from hepatic cells upon stress or nutrient
shortage, as demonstrated by silencing of P2X7 and
P2X4 genes with small interference RNA and treatment
with the P2X inhibitor, A438079 [310]. Recently,
studies on RAW264.7 murine macrophages where
P2X4 was knocked-down with short hairpin RNAs
showed that P2X4 was implicated in the suppression of
P2X7-mediated cell death induced by high ATP or a
P2X7 agonist [311]. Lastly, a reference should be made
to the involvement of hemichannels in purinergic
signaling, in the ATP-induced ATP release. Stress-
opened hemichannels release ATP, an agonist of the
P2X and P2Yreceptors. The P2X2, P2X4 and P2X7
receptors selectively induce cellular penetration of
large molecules, promoting inflammatory stress,
cellular damage and death. Consequently, the cross-
talk between hemichannels and purinergic receptors
may open a therapeutic avenue in various pathologies
including inflammation, atherosclerosis, and chronic
pain [312].
Endoglin, also named TGFβ type III receptor or
CD105, plays a regulatory role in TGFβ signaling
pathways [313]. Endoglin is predominantly expressed
by cells found in the vessel wall, including EC, vascular
SMC and macrophages [314]. While weak or no
expression of endoglin was reported in normal arteries,
strong endoglin expression was found in
atherosclerotic plaques [315, 316]. Two isoforms of
endoglin, long (L-endoglin) and short (S-endoglin),
having different size of the intracellular domain, level of
phosphorylation, affinity to receptor as well as their
capacity to regulate TGFβ-dependent responses, have
been identified [317-319]. The removal of the endoglin
extracellular domain by MMP-14 generates its soluble
form that contributes to the pathogenesis of
preeclampsia, characterized by maternal endothelial
dysfunction and hypertension [320]. The levels of S-
endoglin were found to be increased at early stages of
atherosclerosis, but decreased in later stages of
atherosclerotic process [321]. In senescent cultured
human EC and in vascularized tissues of aged mice,
the S/L ratio of endoglin isoforms was found to be
increased, suggesting that S-endoglin is induced during
endothelial senescence contributing to age-dependent
vascular pathology. Moreover, S-endoglin is important
in NO-dependent vascular homeostasis, as
demonstrated in mice overexpressing S-endoglin in
EC. Interestingly, the long form of endoglin show pro-
angiogenic and atheroprotective effects through
induction of eNOS activity, whereas the short form of
endoglin has anti-angiogenic effects and aggravates
atherogenesis via down-regulation of eNOS level as
well as TGFβ signaling inhibition [322].
In order to ascertain the role of endoglin in
atherogenesis, knockout mice were generated.
Homozygote endoglin deficient mice displayed
embryonic lethality due to defects in vessel and heart
development [323]. Next, mice expressing only one
allele of endoglin, in two different inbred strains,
129/Ola and C57BL/6 were generated as models of
hereditary hemorrhagic telangiectasia [324]. These
haplo-insufficient mice do not develop spontaneous
atherosclerosis, and were not crossed onto athero-
prone mice background till present. On our knowledge,
the effect of endoglin deficiency in atherosclerotic mice
models was not assessed, yet. In apoE-/-LDLR-/- mice
hypercholesterolemia increased plasma soluble
endoglin levels, while it simultaneously decreased its
expression in aorta, together with the decrease of the
athero-protective markers p-Smad2 and VEGF [325].
However, the source and role of endoglin in blood
serum have not been exactly identified. Still, from this
and other studies the level of plasma endoglin seems
to correlate with the progression of atherosclerosis,
allowing treatment efficacy monitoring [326]. Recently,
endoglin has been reported to be a direct receptor for
leucine-rich alpha-2-glycoprotein 1 (LRG1), a novel
Beyond Lipoprotein Receptors Current Molecular Medicine, 2015, Vol. 15, No. 10 17
regulator of angiogenesis [327]. In the presence of
TGF-β1, LRG1 is mitogenic to EC and promotes
angiogenesis via Smad1/5/8 signaling pathway.
Insulin Receptors
Insulin is involved in glucose homeostasis via
increased GLUT4 transporter mediated tissue glucose
uptake. The insulin receptor (IR) is encoded by a single
gene INSR, from which either IR-A or IR-B isoform is
produced through alternative splicing. IGF-I and IGF-II
can also bind the IRs. The IR-A is the fetal form of IR,
mainly involved in mitogenesis upon IGF-II binding,
while the IR-B form is the one implicated in glucose
homeostasis [328]. Together with insulin-like growth
factor receptor and insulin receptor-related receptor, IR
is a member of a receptor tyrosine kinases subfamily,
all sharing a tetrameric α2β2 subunit structure. Insulin
receptors are expressed and active in many cell types
involved in atherosclerosis: EC, vascular SMC,
macrophages. A series of proteins have been identified
as kinase substrates for the insulin receptor, including
the insulin receptor substrate (IRS) proteins, of which
IRS-1 and IRS-2 are the most important for insulin
signaling. Phosphorylated Tyr residues on the IRSs
dock selective downstream signaling molecules. Both
the phosphatidylinositol-3-kinase and MAPK pathway
are involved in insulin signaling [329], the first being
utilized solely by metabolic tissues, while the latter is
associated with survival and proliferation. Insulin
resistance leads to systemic hyperglycemia /
hyperinsulinemia, a state which has now been
recognized to be associated with atherosclerosis. This
effect appears to be LDL-independent as shown in
KKAy murine model of obesity, spontaneous diabetes
and insulin resistance, despite normal LDL level. In this
model, the enhanced monocyte adhesion to EC was
connected to decreased insulin signaling in
macrophages [330]. Similarly, specific IRS-2 deficiency
in macrophages led to their accumulation in the
vascular wall accompanied by increased expression of
proinflammatory mediators [331]. Also, irradiated
LDLR-/- mice developed larger lesions when
transplanted with bone marrow from IR-/- mice
compared with the IR+/+ recipients.
Reduced Akt activity detected in macrophages was
associated with increased apoptosis and formation of
necrotic cores within atherosclerotic plaques [332].
These results were consistent with those obtained in
Akt2-/-LDLR-/- mice, which developed larger and more
complex atherosclerotic lesions than the LDLR-/- mice,
besides impaired glucose tolerance [333]. Moreover,
systemic IRS-2-deficiency mediated insulin resistance-
enhanced atherosclerosis in apoE-/- mice. Defective
insulin signaling accelerated atherosclerosis
independently of glucose, triglyceride, cholesterol,
HDL, VLDL or FFA levels in apoE-/- mice heterozygous
for the IR and the IRS-1. In this case the following
findings were reported: vascular dysfunction through
reduced levels of vascular phospho-eNOS, ameliorated
endothelium-dependent vasorelaxation as well as
increased VCAM-1 expression, SMC proliferation and
inflammation [334]. In contrast, fetal liver cell
transplantation of IRS-2-/-apoE-/- cells alleviated
atherosclerosis in apoE-/- mice [335]. Knocking out the
vascular endothelial IR receptor did not significantly
affect vascular development or glucose homeostasis
under basal metabolic conditions, but reduced the
expression of vasoactive mediators (eNOS, endothelin)
and induced insulin resistance on low-salt diet [336].
Selective endothelial insulin resistance generated by
overexpression of a dominant-negative mutant human
IR exhibited endothelial dysfunction, decreased NO
bioavailability and increased generation of ROS,
independent of a significantly altered metabolic
phenotype [337]. Knockout mice studies showed that
IRS-2 delays neointima development in insulin-
resistant states [338]. Overall, most studies of impaired
insulin signaling via its receptor and receptor
substrates point toward IR as an important anti-
atherogenic factor.
Atherosclerosis and its complications represent the
leading cause of mortality worldwide, despite
tremendous advances over the last years.
Experimental atherosclerosis studies using animal
models successfully showed the reduction or
regression of the atheroma. Nonetheless, the
extrapolation of the findings to atherosclerosis in
humans is often difficult due to major differences in
plaque morphology, plaque cholesterol content, or
absence of fibrous caps. However, it is noteworthy that
the elucidation of the molecular mechanisms involved
in the initiation and progression of atherosclerotic
lesions revealed new anti-atherosclerotic targets that
can be used to develop novel or complementary
strategies to overcome the barriers imposed by the
conventional treatments. There is no doubt that
additional partakers that either protect against or
enhance atherosclerosis will continue to be identified.
The differential expression of the receptors in
pathological compared to physiological states opens
various opportunities in the design of new therapeutic
approaches for atherosclerosis. Thus, we discussed
herein the main non-lipoprotein receptors, not related
to the lipid metabolism, used as targets for atheroma
regression, as overviewed in Table 1. Their cellular
distribution and their positive or negative effects in the
atheromatous plaque are schematically illustrated in
Fig. (1). Atherosclerosis is a tremendously complex
process regarding the simultaneous dysregulation of
multiple pathways in various cell types. We have only
focused on the main receptors described in the
literature as potential targets, based on murine
knockout studies. Despite the accumulation of
remarkable data which enhanced our general
knowledge regarding atherosclerosis, certain reports
appear controversial and rather puzzling. We are
aware of the fact that we could not discuss all the
controversies regarding the receptors described,
unless we pass beyond the scope of this review. These
aspects refer to processes related to endogenous
versus exogenous ligand binding, stoichiometry of
18 Current Molecular Medicine, 2015, Vol. 15, No. 10 Trusca et al.
binding, the alternative homo-/hetero- oligomerization,
desensitization/down-regulation of the receptors,
different pathways of receptor internalization, the
presence of isoforms (long, short, soluble), the
presence of pro- or anti-atherogenic single nucleotide
polymorphisms, the existence of divergent signaling
Considering the permanent cross talk between the
metabolic and the inflammatory pathways, care should
be given when assessing the amount of individual
molecules, since the ratio of various species rather
than the particular concentration should make a differ-
ence in the evolution of the dysfunction. It is expected
that future studies will clarify how atherogenesis is
affected by various factors not sufficiently taken into
account so far.
Taken together, drugs targeted towards the cellular
receptors that play an important role in atherosclerosis,
may be included in novel treatment strategies, which
should include more than a single therapeutic target.
ATF = Activating Transcription Factor
AP-1 = Activator Protein 1
ACAT = Acyl-coenzyme A:cholesterol
AMPK = AMP-activated protein kinase
Ang = Angiopoietin
AT = Angiotensin
apoE = Apolipoprotein E
ABCG1 = ATP binding cassette transporter G1
ABCA1 = ATP-binding cassette transporter 1
BMDC = Bone Marrow Derived Cells
CB = Cannabinoid
CETP = Cholesteryl Ester Transfer Protein
JNK = c-Jun N-terminal kinase
DC = Dendritic cell
ECs = Endothelial Cells
eNOS = Endothelial Nitric Oxide Synthase
ET = Endothelin
FGF = Fibroblast Growth Factor
IGF = Insulin-like Growth Factor
IR = Insulin Receptor
LDLR = LDL receptor
LCAT = Lecithin-cholesterol acyltransferase
LXR = Liver X Receptor
MMP = Matrix metalloproteinase
NOS = Nitric Oxide Synthase
PPAR = Peroxisome Proliferator-Activated
PDGF = Platelet-Derived Growth Factor
FcγRs = Receptors for the Fc region of IgG
ROS = Reactive Oxygen Species
RXR = Retinoid X Receptor
SMC = Smooth Muscle Cells
TGF-β = Transforming Growth Factor-β
TP = Thromboxane receptor
TLR = Toll-like receptor
TNF = Tumor Necrosis Factor
Trk = Tyrosine receptor kinase
VEGF = Vascular Endothelial Growth Factor.
The authors confirm that this article content has no
conflict of interest.
This work was supported by a grant of the
Romanian National Authority for Scientific Research,
CNCS UEFISCDI, project number PN-II-ID-PCE-
2011-3-0591 (grant awarded to AG), and by the
Romanian Academy. Violeta Trusca acknowledges the
support of the strategic grant POSDRU/159/1.5/S/
133391-financed by the European Social Found within
the Sectorial Operational Program Human Resources
Development 2007 2013. The authors would like to
thank Dr. Ovidiu Croitoru for graphic design.
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