Clinica Chimica Acta 428 (2014) 1–8

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Apelin and its receptor APJ in cardiovascular diseases

Xiao-Hua Yu a, Zhi-Bin Tang c, Li-Jing Liu d, Hong Qian d, Shi-Lin Tang c, Da-Wei Zhang e,
Guo-Ping Tian f,1, Chao-Ke Tang a,b,2
a
Life Science Research Center, University of South China, Hengyang, Hunan 421001, China
b
Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang, Hunan 421001, China
c
Department of Orthopedics, The First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China
d
Department of Clinical Medicine, Huaihua Medical College, Huaihua, Hunan 418000, China
e
Department of Pediatrics and Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
f
Department of Cardiovascular Medicine, The Second Affiliated Hospital, University of South China, Hengyang 421001, Hunan, China

a r t i c l e i n f o a b s t r a c t

Article history: Apelin is an adipokine that has been identified as an endogenous ligand for the orphan receptor APJ. Apelin and
Received 6 July 2013 APJ are expressed in a diverse range of tissues with particular preponderance for the heart and vasculature.
Received in revised form 31 August 2013 Apelin has powerful positive inotropic actions and causes endothelium- and nitric oxide-dependent vasodilata-
Accepted 1 September 2013
tion. Growing evidence shows that apelin/APJ system functions as a critical mediator of cardiovascular homeosta-
Available online 18 September 2013
sis and is involved in the pathophysiology of cardiovascular diseases. Targeting apelin/APJ axis produces
Keywords:
protection against cardiovascular diseases. In the current review we have summarized recent data concerning
Apelin the role and therapeutic potential of apelin/APJ in several major cardiovascular diseases. An increased under-
APJ standing of the cardiovascular actions of apelin/APJ system will help to develop novel therapeutic interventions
Atherosclerosis for cardiovascular diseases.
CAD © 2013 Elsevier B.V. All rights reserved.
Heart failure
Hypertension

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Regulation of apelin and APJ expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Apelin/APJ cellular signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4. Cardiovascular physiological actions of apelin and APJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
5. Roles of apelin and APJ in cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
5.1. Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
5.2. CAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
5.3. Heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.4. Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.5. PAH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.6. MIRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.7. Atrial fibrillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6. Therapeutic potential of apelin/APJ in cardiovascular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Abbreviations: Ang II, angiotensin II; CAD, coronary heart disease; PAH, pulmonary arterial hypertension; MIRI, myocardial ischemia–reperfusion injury; HIF-1α, hypoxia-inducible factor
1α; TGF-β, transforming growth factor-β; BMP, bone morphogenic protein; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; MCP-1, monocyte
chemoattractant protein-1; PI3K, phosphatidylinositol 3-kinase; TNF-α, tumor necrosis factor-α; JNK, c-Jun N-terminal kinase; VSMC, vascular smooth muscle cell; IRS1, insulin receptor
substrate 1; GSK, glycogen synthase kinase; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; HDAC, histone deacetylase; MEF2, myocyte enhancer factor 2; Ang-1, angiopoietin-1; VEGFA,
vascular endothelial growth factor type A; PDGF-BB, platelet-derived growth factor-BB; NO, nitric oxide; ACAT1, acyl coenzyme A:cholesterol acyltransferase-1; ABCA1, ATP-binding cassette
transporter A1; apo E, apolipoprotein E; CAD, coronary heart disease; FGF2, fibroblast growth factor 2; PPREs, PPAR response elements; PKB, protein kinase B; PKC, protein kinase C; TGF-β,
transforming growth factor-β; BNP, brain natriuretic peptide.
E-mail addresses: pgtian555@hotmail.com (G.-P. Tian), tangchaoke@qq.com (C.-K. Tang).
1
Department of Cardiovascular Medicine, Second Affiliated Hospital of University of South China, Hengyang 421001, Hunan, China. Tel./fax: +86 734 8281852.
2
Institute of Cardiovascular Research, University of South China, Hengyang, Hunan 421001, China. Tel./fax: +86 734 8281853.

0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cca.2013.09.001
2 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 1–8

7. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Introduction
APJ, first identified from a human genomic library in 1993, belongs
to a member of seven trans-membrane G protein-coupled receptor fam-
ily. Its amino acid sequence has 31% homology with that of the human
angiotensin II (Ang II) type 1 receptor, but it cannot bind Ang II. Of
note, APJ has no subtypes and is the only known receptor of apelin, an
endogenous peptide extracted from bovine stomach in 1998. Apelin is
produced as a 77 amino acid preproapelin which is cleaved by an
angiotensin-converting enzyme to form several shorter C-terminal bio-
active peptides, such as apelin-13, -16, -17, -19, -36 as well as the
pyroglutamate form of apelin-13, [Pyr1]apelin-13 [1]. It has been sug-
gested that apelin-13 and apelin-17 possess much stronger activity
than apelin-36. Apelin and APJ constitute a signaling pathway and are
widely expressed in various tissues, especially in the cardiovascular sys-
tem including the heart, kidney and vessels [2]. Abundant evidence re-
veals that apelin/APJ pathway is a critical regulator of cardiovascular
function [3,4] and plays an important role in the occurrence and devel-
opment of cardiovascular diseases including atherosclerosis, coronary
heart disease (CAD), heart failure, hypertension, pulmonary arterial hy-
pertension (PAH), myocardial ischemia–reperfusion injury (MIRI) and
atrial fibrillation [5]. Targeting apelin/APJ axis is protective against
these diseases, one of the major causes of death in the world [6]. Thus,
we review the regulation, cellular signaling pathways and cardiovascu-
lar physiological actions of apelin/APJ system and explore its emerging
pathogenetic significance and therapeutic potential in several major
cardiovascular diseases.
2. Regulation of apelin and APJ expression
The expression of apelin and APJ is regulated by multiple factors.
Geurts et al. reported that endocannabinoid in combination with lipo-
polysaccharide markedly upregulate apelin and APJ mRNA expression
in the adipose tissue of obese and diabetic mice [7]. Mechanistically,
cannabinoid receptor, interleukin (IL)-1 and tumor necrosis factor-α
(TNF-α) are involved in this effect [7]. Hypoxia induces apelin expres-
sion in human adipocytes through a hypoxia-inducible factor 1α (HIF-
1α)-dependent mechanism [8], whereas reoxygenation after hypoxia
decreases its expression in human endothelial cells by activating fatty
acid transport protein [9]. Interestingly, hypoxia, TNF-α and Ang II sig-
nificantly enhance the expression of APJ in HepG2 cells [10]. Ang II
type 1 receptor can stimulate apelin and APJ secretion through the activa-
tion of phospholipase C (PLC)-β/inositol-1,4,5-trisphosphate (IP3)/Ca2+/
protein kinase C (PKC)/MAPK kinase/extracellular signal-regulated pro-
tein kinase 1/2 (ERK1/2) and PLC-β/diacylglycerol (DAG)/PKC ways,
whereas Ang II type 2 receptor can reduce apelin and APJ production
through the inhibition of adenylyl cyclase/cAMP/PKA and guanylate
cyclase/cGMP/PKG pathways in 3T3-L1 adipocytes, indicating a differ-
entially regulatory manner of apelin/APJ expression by Ang II type 1
and 2 receptors [11]. Bone morphogenic proteins (BMPs) are a sub-
group of the transforming growth factor-β (TGF-β) superfamily. Some
BMPs such as BMP4, 7 and 9 have been shown to diminish apelin
mRNA levels in endothelial cells by stimulating BMP receptor 2 and
then increasing expression of Smad 7, a transcriptional repressor of
apelin [12]. Obesity surgery-induced weight loss leads to a significant
reduction in apelin expression in omental and subcutaneous adipose
tissue, contributing to improved insulin sensitivity [13]. Additionally,
preterm and term spontaneous labors downregulate apelin expression
but have no effect on APJ expression in human primary amnion cells
[14]. However, the involved mechanisms are unclear.
3. Apelin/APJ cellular signaling pathways
Apelin/APJ triggers a variety of cellular signaling pathways (Fig. 1).
Recent studies from our laboratory showed that apelin-13 induces vas-
cular smooth muscle cell (VSMC) proliferation by the upregulation of
Cyclin D1 expression, which is involved in an ERK-dependent activation
of Jagged-1/Notch3 signaling [15]. Lu and colleagues reported that
apelin/APJ increases the expression of intercellular adhesion molecule-
1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte
chemoattractant protein-1 (MCP-1) in human umbilical vein endotheli-
al cells by promoting nuclear translocation of nuclear factor kappa-κB
(NF-κB) and then increasing phosphorylation of c-Jun N-terminal ki-
nase (JNK) via a pertussis toxin (PTX)-sensitive G-Protein [16]. In con-
trast with this report, apelin inhibits MCP-1 expression in endothelial
cells through activation of the phosphatidylinositol 3-kinase (PI3K)/
Akt pathway, followed by suppression of Smad3 [17]. This discrepancy
may be due to different experimental protocols and apelin isoforms. It
has been suggested that apelin ameliorates TNF-α-induced reduction
of glycogen synthesis in HepG2 cells by improving the JNK/insulin
receptor substrate 1 (IRS1)/glycogen synthase kinase (GSK) signaling
pathway, indicating the anti-insulin resistance properties of apelin
[18]. Knockdown of apelin expression in primary amnion cells using
siRNA significantly increases IL-1β-induced IL-6 and IL-8 secretion as
well as cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE2)
release [14]. Apelin/APJ system inhibits histone deacetylase (HDAC) 4
and HDAC5 phosphorylation and subsequently upregulates myocyte
enhancer factor 2 (MEF2) expression, leading to Kruppel-like factor 2
(KLF2) transcription in endothelial cells [19]. Than et al. have demon-
strated that apelin interaction with APJ receptor suppresses lipolysis in
mature adipocytes, and the mechanisms are associated with AMP
kinase (AMPK) dependent enhancement of perilipin expression [20].
Activation of APJ has been shown to stimulate angiopoietin-1 (Ang-1)
expression, which in turn triggers the synthesis of vascular endothelial
growth factor type A (VEGFA) and platelet-derived growth factor-BB
(PDGF-BB) in HepG2 cells [10]. In addition, treatment with apelin
can significantly promote Fas-induced hepatocyte apoptosis at least
partially via JNK activation [21]. However, Cui and colleagues found
that apelin markedly inhibits serum deprivation-induced apoptosis of
human VSMCs, and the anti-apoptotic action is mediated through
stimulating PI3K/Akt signaling dependent increase of Bcl-2 protein
expression and decrease of Bax protein expression [22]. Many
more apelin/APJ-regulated cellular signaling pathways will be prob-
ably discovered in future studies.
4. Cardiovascular physiological actions of apelin and APJ
Apelin/APJ system has a number of physiological actions such as fluid
homoeostasis, glucose homeostasis, feeding behavior and immunity [23].
Several lines of evidence demonstrate that the cardiovascular system is
the main target of apelin and its receptor APJ (Fig. 2). Apelin has been
reported to lower arterial blood pressure through a nitric oxide (NO)-de-
pendent mechanism [24], which is abrogated in APJ-deficient mice [25].
In addition, administration of apelin causes a concentration-dependent
vasodilatation in endothelium-intact mammary artery but has no
effect after endothelial removal, suggesting an endothelium-dependent
 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 1–8 3

Fig. 1. Schematic of apelin/APJ-triggered intracellular signal transduction pathways. Apelin can induce VSMC proliferation by activating ERK/Jagged-1/Notch3/Cyclin D1 signaling pathway.
Apelin inhibits TNF-α-induced reduction of glycogen synthesis through improving JNK/IRS1/GSK signaling in HepG2 cells. Apelin promotes Fas-induced hepatocyte apoptosis in a
JNK-dependent mechanism but prevents human VSMC apoptosis via PI3K/Akt-induced increase in the ratio of Bcl-2 to Bax. Apelin/APJ significantly increases the expression of ICAM-1,
VCAM-1 and MCP-1 in human umbilical vein endothelial cells through G-Protein/NF-κB/JNK pathway yet reduces MCP-1 expression in endothelial cells through activation of PI3K/Akt/
Smad3. Treatment of primary amnion cells with apelin decreases IL-1β-induced IL-6 and IL-8 secretion as well as COX-2-mediated PGE2 release. The apelin/APJ axis stimulates KLF2 transcrip-
tion in endothelial cells through inhibition of HDAC 4 and HDAC5 phosphorylation and subsequent upregulation of MEF2 expression. Apelin also suppresses lipolysis of mature adipocytes via
AMPK-dependent enhancement of perilipin levels. Additionally, apelin increases the synthesis of VEGFA and PDGF-BB by inducing Ang-1 expression in HepG2 cells. P: phosphorylation.

hypotensive effect of apelin [26]. Injection of apelin into the ischemic
myocardium facilitates neovascularization in the peri-infarct area
through paracrine activity [27]. Treatment with apelin also decreases sys-
temic venous tone [28], and inhibits arginine vasopressin release to pro-
mote diuresis [29]. On the other hand, intraperitoneal injection of apelin
reduces left ventricular preload and afterload without causing cardiac hy-
pertrophy [30]. In a study of isolated perfused rat hearts, apelin is identi-
fied as one of the most potent endogenous positive inotropic substances
[31]. Moreover, isolated left ventricular cardiomyocytes lacking either
apelin or APJ display less sarcomeric shortening and impaired velocity
of contraction and relaxation with no difference in calcium transient
[32]. These findings indicate an important role for apelin/APJ system in
maintaining basal cardiac function.
Fig. 2. Cardiovascular physiological actions of apelin/APJ system.
5. Roles of apelin and APJ in cardiovascular diseases
5.1. Atherosclerosis
Atherosclerosis is a complex, chronic inflammatory disease of the
vessel wall with lipid-laden lesions. Formation of macrophage foam
cells in the intima is a major hallmark of early stage atherosclerotic
lesions. Uncontrolled uptake of oxidized low-density lipoprotein
(ox-LDL) via CD36, excessive cholesterol esterification by acyl coen-
zyme A:cholesterol acyltransferase 1 (ACAT1) and/or impaired choles-
terol efflux mediated by ATP-binding cassette transporter A1 (ABCA1)
result in the accumulation of cellular cholesterol ester, which subse-
quently triggers the formation of foam cells [33]. Studies from our
laboratory revealed that apelin-13, the predominant apelin isoform in
circulatory system, significantly promotes intracellular cholesterol ef-
flux and reduces macrophage foam cell formation by increasing
ABCA1 protein levels, indicating an potential antiatherogenic function
of apelin-13 [34]. Mechanistically, the regulation of ABCA1 expression
by apelin-13 is through activating the PKCα pathway and inhibiting
calpain activity [34]. However, it remains unclear whether apelin mod-
ulates CD36 and ACAT1 expression. The influence of apelin on lipid me-
tabolism may become an important research topic in the future.
The precise role of apelin and APJ in protecting against the develop-
ment of atherosclerosis remains uncertain (Table 1). Rittig et al. ob-
served that plasma apelin levels are not associated with early stages of
atherosclerosis in young subjects prone to atherosclerosis and type 2 di-
abetes [35]. Nevertheless, Kadoglou and colleagues have demonstrated
that patients with carotid atherosclerosis show lower apelin levels as
compared to healthy controls, and apelin increment is independently as-
sociated with the atorvastatin-induced increase of gray-scale median
4 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 1–8

Table 1
Effects of apelin/APJ system on cardiovascular diseases.

Disease types Experiment models Pathway Action Ref.

Atherosclerosis THP-1 macrophage-derived foam cells ↑PKCα/ABCA1, ↓calpain Protection [34]

Human plasma ? No [35]
Human plasma ? Protection [36,37]
Mice in vivo ↑NO, ↓Ang II Protection [38]
Mice in vivo knockout ↓Superoxide radicals, ↓NADPO Protection [39]
Human in vivo ? Promotion [40,41]
CAD Human in vivo (gene studies) ? Protection [42]
Human plasma ? Protection [43–48]
Rats in vivo (coronary ligation) ↓oxidative injury, ↑NO Protection [49]
Mice in vivo (coronary ligation) ↑SDF-1α/CXCR-4, ↑PCs Protection [50]
Rats in vivo (coronary ligation) ? Protection [51]
Rats in vivo (coronary occlusion) ? Protection [52]
Heart failure Rats in vivo ? Protection [53]
Mice in vivo knockout ↑Systolic dysfunction Promotion [54]
Mice in vivo knockout ↓β-arrestins Protection [55]
Human in vivo ? Protection [56,57]
Human plasma ? Protection [58,59]
Mice in vivo/in vitro (cardiac fibroblasts) ↓TGF-β, ↓SphK1 Protection [62]
Human in vivo ↑Vasodilatation, ↑LVEF Protection [63,66]
Rats in vivo ↓Cardiac dysfunction Protection [64,65]
Hypertension Human plasma ? Protection [67–69]
Human in vivo ? Protection [70]
Human in vivo (gene studies) ? Protection [71]
Rats in vivo ↓RAS Protection [72,73]
Rats ex vivo (aorta) ↑L-Arg/NOS/NO Protection [74]
Rats in vivo ? Promotion [75]
Mice in vivo ? Promotion [76]
PAH Human plasma/mice in vivo knockout ↓AMPK, ↓KLF2/eNOS Promotion [77]
Human in vitro (PAECs)/mice in vivo ? Protection [78]
Rats in vivo ↓BNP, ↓AGT, ↓ET-1 Protection [79]
Human in vitro (PAECs)/mice in vivo ↑miRNA/FGF2 Protection [80]
MIRI Rats ex vivo (hearts) ? Protection [82]
Rats in vivo ↑PI3K/Akt, ↑AMPK, ↑ERK Protection [83]
Rats ex vivo (hearts)/myocardial cells ↓ROS, ↓MDA, ↓LDH Protection [84]
Mice/rats in vivo/myocardial cells ↑RISK, ↓MPTP opening Protection [85]
Atrial fibrillation Human plasma ? Protection [87,88,90]

↑ indicates activation or promotion. ↓ indicates inhibition.

score, a valid index of carotid plaque vulnerability, suggesting that
the atorvastatin-induced modification of apelin may beneficially affect
carotid plaque stability [36]. Aerobic training has been shown to increase
plasma apelin levels and attenuate carotid intima–media thickness
progression in patients with type 2 diabetes [37]. Ang II is a potent
proatherosclerotic factor. One recent study showed that apelin treatment
abrogates Ang II-induced atherosclerosis and abdominal aortic aneurysm
formation in apolipoprotein E (apo E) knockout mice by promoting NO
production and inhibiting Ang II cellular signaling [38]. On the other
hand, the apelin/APJ system is a mediator of oxidative stress in vascular
tissue. APJ and apo E double-knockout (APJ−/−apoE−/−) mice fed a
high-cholesterol diet show a reduction in atherosclerotic lesions, vascular
production of superoxide radicals and the expression of nicotinamide-
adenine dinucleotide phosphate oxidase (NADPO) subunits when
compared with APJ+/+apoE−/− mice, revealing a potential role of APJ in
the promotion of oxidative stress-linked atherosclerosis [39]. Apelin is
upregulated in human atherosclerotic coronary artery, colocalizes to the
plaque with markers for macrophages and smooth muscle cells and
potently constricts human coronary artery [40]. In addition, the levels of
apelin and its receptor APJ expression are increased in patients with calci-
fied aortic valve stenosis, which is an actively regulated pathobiological
process with some hallmarks of atherosclerosis [41]. Collectively, the im-
pact of apelin/APJ system on the occurrence and development of athero-
sclerosis is complex and needs to be further investigated.
5.2. CAD
The role of apelin and APJ in the pathogenesis of CAD has obtained a
great attention (Table 1). Recent data by Jin et al. show that interactive
effects of genetic defects in apelin/APJ pathway may confer a potential
risk for CAD in Chinese hypertensive patients [42]. It has been reported
that plasma apelin levels are reduced early after acute myocardial in-
farction, elevated significantly over time, but still remained significantly
lower than in the healthy control population at 24 weeks [43,44]. Re-
duced plasma levels of apelin 36 are also found in patients with first
ST-elevation myocardial infarction during the first five days, and this re-
duction is independent on the degree of left ventricular dysfunction and
prognosis [45,46]. In a KOZANI study, apelin concentrations are consid-
erably lower in CAD patients in comparison with healthy controls. The
plasma levels of apelin are even lower in unstable angina and acute
myocardial infarction groups on admission as compared to asymptom-
atic CAD group, and apelin levels are negatively correlated with the se-
verity and the acute phase of CAD, suggesting its involvement in the
progression and destabilization of coronary atherosclerotic plaques
[47]. Additionally, decreased apelin levels are observed in subjects
with stable angina and the plasma apelin levels are negatively correlat-
ed with the severity of coronary stenosis [48]. These data suggest that
plasma apelin level may be a diagnostic indicator for CAD patients.
The therapeutic effects of apelin in the management of CAD have
been examined. Azizi et al. demonstrated that post-infarct treatment
with [Pyr1]-apelin-13 significantly attenuates myocardial damage
through the reduction of oxidative injury and enhancement of NO pro-
duction in the rat model of myocardial infarction [49]. In addition,
apelin-13 has been found to promote angiogenesis and ameliorate car-
diac repair postmyocardial infarction by a mechanism involving the
upregulation of stromal cell-derived factor-1α (SDF-1α)/CXC chemo-
kine receptor-4 (CXCR-4) and homing of vascular progenitor cells
(PCs) [50]. Injection of apelin-12 to experimental animals also limits
the myocardial infarction size and reduces damage to cardiomyocyte
membrane [51]. Additionally, angiopoietin-1 exerts its beneficial effect
 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 1–8
in the repair of diabetic mouse infarcted hearts through upregulation of
apelin expression [52]. However, apelin protection against CAD has not
been tested in humans. This promising effect of apelin can be applied in
humans in the conditions that myocardial infarction severely impairs
the heart function, the most common cause of heart failure. Application
of apelin during the percutaneous coronary intervention may have ther-
apeutic benefits as well.
5.3. Heart failure
There is increasing interest about the role of apelin and its receptor
APJ in heart failure development (Table 1). In murine model of hyper-
tensive heart disease, the expression of cardiac apelin and APJ shows
no change at the stage of compensatory left ventricular hypertrophy
but is markedly decreased at the stage of heart failure [53]. Apelin-
deficient mice develop progressive heart failure associated with systolic
dysfunction [54], but mice lacking APJ confer resistance to heart failure
by inhibiting the expression of β-arrestins, suggesting an apelin-
independent function of APJ [55]. In humans, myocardial apelin and
APJ expression is downregulated in subjects with heart failure [56,57].
In addition, several groups have reported that plasma apelin levels are
significantly lower in patients with heart failure compared with age-
matched controls, suggesting that reduced levels of plasma apelin may
act as a reliable biomarker for detecting heart failure [58,59]. Recently,
a variety of mechanisms have been proposed to explain how apelin re-
duction causes heart failure. Apelin can lower left ventricular preload
and afterload in mice [30], and has strong positive inotropic effects
[31,60]. In anesthetized rats, microinjection of apelin into subfornical
organ reduces blood pressure and heart rate [61]. Activation of cardiac
fibroblasts and their differentiation into myofibroblasts are critical
events in the progression of cardiac fibrosis that results in end-stage
heart failure. Pchejetski et al. showed that apelin prevents TGF-
β-induced activation of cardiac fibroblasts and collagen production by de-
creasing sphingosine kinase 1 (SphK1) activity [62]. They also demon-
strated that apelin administration during the phase of reactive fibrosis
inhibits structural remodeling of the myocardium and ventricular dys-
function, revealing a novel link between cardiac fibrosis and heart failure
[62]. However, more mechanistic studies are necessary to elucidate the
effects of apelin/APJ system on the pathology of heart failure.
Based on the cardioprotective actions of apelin and APJ, strategies to
enhance apelin signaling may therefore help to retard the progression of
heart failure. It has been reported that acute apelin administration
causes peripheral and coronary vasodilatation and increases cardiac
output in patients with chronic heart failure [63]. Ang II receptor blocker
olmesartan and exogenous apelin-13 significantly ameliorate cardiac
dysfunction and remodeling in Dahl salt-sensitive hypertensive rats
with end-stage heart failure through restoring apelin and APJ expres-
sion [64,65]. Intracoronary implantation of bone marrow mononuclear
cells into ischemic hearts increases apelin levels and thereby contrib-
utes to the improvement of cardiac function in patients with severe
heart failure [59]. In another study, prolonged 6-hour [Pyr1]apelin-13
infusion leads to a sustained increase in cardiac index with increased
left ventricular ejection fraction (LVEF) in patients with chronic heart
failure, suggesting that APJ agonism may possess a major promise to
complement current optimal medical therapy in chronic heart failure
patients [66].
5.4. Hypertension
Recently, a variety of studies have focused on the association of
apelin/APJ system with hypertension (Table 1). Masked hypertension
is a significant predictor of cardiovascular diseases, and plasma apelin
levels are significantly lower in the masked hypertensive group as com-
pared to normotensive controls [67]. In patients with essential hyper-
tension, circulating apelin levels are also reduced and independently
associated with more profound left ventricular systolic and diastolic
5
function impairment [68]. In addition, hypertensive rats show reduced
plasma apelin levels and downregulation of APJ in kidneys [69]. Notably,
APJ expression is remarkably downregulated in early-onset pregnancy-
induced hypertension placentas, reflecting an aggravated placental
condition with poor fetal growth [70]. The G212A allele of APJ exerts a
protective effect against hypertension development [71]. It has been
reported that apelin reduces blood pressure in deoxycorticosterone
acetate-salt-induced hypertensive rats by inhibiting renin–angiotensin
system (RAS) [72,73]. Jia and co-workers observed that apelin-induced
vasodilation is directly associated with the activation of L-arginine
(L-Arg)/nitric oxide synthase (NOS)/NO pathway in the incubated, isolat-
ed rat aorta [74]. On the other hand, the expression of apelin is significant-
ly enhanced in the rostral ventrolateral medulla (RVLM) of spontaneously
hypertensive rat when compared with normotensive rats [75]. Direct mi-
croinjection of exogenous apelin-13 into RVLM leads to chronic blood
pressure elevation and cardiac hypertrophy in normotensive rats [75].
NG-nitro-L-arginine methyl ester (L-NAME), a potent endothelial NOS
(eNOS) inhibitor, can induce hypertension. Apelin can transiently elevate
blood pressure in L-NAME-treated mice although it decreases blood pres-
sure in non-treated mice, indicating that apelin functions as a vasopressor
peptide under pathological conditions [76]. Together, apelin may be used
as a therapeutic agent in the treatment of hypertension in the future
while we should pay attention to the complexity of regulation of blood
pressure by apelin. Further researches are required to determine the pre-
cise role for apelin/APJ signaling in controlling blood pressure.
5.5. PAH
PAH is a severe disease characterized by pulmonary vasoconstric-
tion, pulmonary arterial remodeling, abnormal angiogenesis and im-
paired right ventricular function. Apelin and its receptor APJ are highly
expressed in the pulmonary vasculature (Table 1). Chandra et al. report-
ed that serum apelin levels of patients with pulmonary arterial hyper-
tension are significantly decreased as compared to healthy controls,
and apelin-null mice develop more severe pulmonary arterial hyperten-
sion due to reduced activation of AMPK and Kruppel-like factor 2
(KLF2)/eNOS [77]. Apelin expression is also downregulated in the pul-
monary arterial endothelial cells (PAECs) of the patients with pulmo-
nary arterial hypertension, and administration of apelin reverses PAH
in mice with reduced production of apelin [78]. [Pyr1]-apelin-13 admin-
istration has been shown to inhibit activation of several vasoconstrictors
including type B natriuretic peptide (BNP), angiotensinogen (AGT) and
endothelin-1 (ET-1) and thus retards the progression of right ventricu-
lar hypertrophy and diastolic dysfunction in rats with monocrotaline-
induced pulmonary arterial hypertension [79]. Apelin deficiency leads
to the hyperproliferation of PAECs and VSMCs via increased fibroblast
growth factor 2 (FGF2) levels as a consequence of decreased expression
of miR-424 and miR-503 that directly target FGF2 and downregulate its
expression [80]. Reconstitution of miR-424 and miR-503 in vivo improves
pulmonary arterial hypertension in experimental models, suggesting
that apelin-dependent miRNA/FGF2 signaling axis plays a key role in
the maintenance of pulmonary vascular homeostasis [80].
5.6. MIRI
Apelin and APJ also involve in MIRI (Table 1) [81]. A recent study
suggests that apelin mRNA is upregulated in ventricular myocardium
from isolated rat hearts undergoing ischemia alone but markedly
decreased after 30 min reperfusion while APJ mRNA levels have no
change, and apelin administered during reperfusion significantly de-
creases infarct size in hearts subject to temporary coronary occlusion
followed by reperfusion [82]. Administration of apelin-13 protects rat
hearts against ischemia–reperfusion injury through inhibiting endo-
plasmic reticulum-dependent apoptosis via activation of PI3K/Akt,
AMPK, and ERK [83]. Apelin treatment also significantly inhibits MIRI
in rats through preventing the generation of reactive oxygen species
6 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 1–8
(ROS), malonaldehyde (MDA) content and lactate dehydrogenase
(LDH) leakage [84]. Studies from Smith laboratory show that apelin-
13 and apelin-36 exhibit direct cardioprotective activity against ische-
mia–reperfusion injury by activating reperfusion injury salvage kinase
(RISK) pathway and delaying mitochondrial permeability transition
pore (MPTP) opening [85]. Collectively, apelin/APJ has protective effects
in myocardial ischemia–reperfusion injury and may constitute an im-
portant therapy target.
5.7. Atrial fibrillation
Atrial fibrillation is one of the most common arrhythmias among
cardiovascular disease patients. Apelin has increasingly been regarded
as an important regulator of cardiovascular homeostasis with direct ef-
fects on cardiomyocyte contractility and electrophysiology (Table 1)
[86]. Ellinor et al. reported that plasma levels of apelin are significantly
lower in subjects with lone atrial fibrillation when compared with con-
trols, representing a possible underlying diathesis predisposing to this
common arrhythmia [87]. A recent study revealed that patients with
plasma apelin levels below the median have a hazard ratio of 3.1 of atrial
fibrillation recurrence with respect to those with high apelin levels [88].
Since brain natriuretic peptide (BNP) is one of the good biomarkers for
atrial fibrillation [89], the authors also found that subjects with both low
apelin and enhanced BNP possess a worse prognosis as compared to
those with either low apelin or enhanced BNP alone [88]. Moreover, car-
dioversion of atrial fibrillation to sinus rhythm resulted in an increase of
apelin and a decrease of BNP levels, whereas the patients who develop
atrial fibrillation recurrence by the end of the follow-up period have
similar values of apelin and BNP on final and initial evaluations [90].
Thus, low plasma apelin levels are an independent predictor for the re-
currence and prognosis of atrial fibrillation. In addition to BNP, apelin
may be of particular value for the identification of high-risk patients.
6. Therapeutic potential of apelin/APJ in cardiovascular diseases
Since the apelin/APJ system plays a key role in the occurrence and de-
velopment of cardiovascular diseases, it seems logical to presume that
targeting this axis should be feasible and represent a new class of poten-
tial therapeutic agents. In agreement, exogenous administration of apelin
significantly reduces LV end-diastolic volume and end-systolic volume as
well as elevates LVEF in dogs with advanced heart failure [91]. High-dose
of atorvastatin has been shown to attenuate early carotid atherosclerosis
in patients with type 2 diabetes by upregulation of apelin expression [92].
Treatment with embryonic stem cells induces recruitment of vascular PCs
in infarcted hearts, thereby improving repair postmyocardial infarction
[50,93]. Given the short circulating half-life of apelin peptide, apelin ana-
logs or modification must be developed to avoid this limitation. Recently,
Iturrioz et al. synthesized the first non-peptidic agonist of apelin receptor
APJ, namely E339-3D6, which shows a 90 nM affinity and serves as a
partial agonist for cAMP production and a full agonist for APJ internaliza-
tion [94]. This compound can induce the vasorelaxation of rat aorta
precontracted with norepinephrine, and potently suppress systemic va-
sopressin release in water-deprived mice when intracerebroventricularly
injected [94]. Wang et al. also designed two novel apelin analogs resistant
to angiotensin converting enzyme 2 cleavage and identified one analog,
which mimicked the function of apelin [95]. They found that the apelin
analog is protective against ex vivo and in vivo MIRI resulting from greater
activation of survival pathways and promotion of angiogenesis [95]. Also,
structural analogs of apelin-12 can limit myocardial infarction [96]. In
addition, Jia and colleagues have successfully produced a 40 kDa polyeth-
ylene glycol (PEG) conjugated apelin-36 (PEG-apelin-36), which is char-
acterized by N-terminal conjugation, high purity (N98%), minimum
reduction of APJ binding affinity and a prolonged circulating life [97].
Similar increases in cardiac EF are observed during the infusion of PEG-
apelin-36 and apelin-36 in normal rats, whereas animals that received
PEG-apelin-36 maintain significantly increased EF over the 100 min
post infusion monitoring period compared with apelin-36-treated
animals; EF increases observed with PEG-apelin-36 and apelin-36 are
greater in the myocardial infarction rats [97]. However, the roles of APJ
agonism and apelin PEGylation need to be assessed in vivo in human. Of
note, apelin has the ability to enhance gastric acid secretion as well [98],
and may represent an important side effect that could have implications
for its tolerability in cardiovascular disease patients.
7. Conclusion and future directions
The discovery of the apelin/APJ axis is an exciting affair in cardiovas-
cular research. Experimental and clinical investigations performed both
in vivo and in vitro have suggested apelin/APJ as a critical mediator of
cardiovascular homeostasis involved in the pathogenesis of several
main cardiovascular diseases including atherosclerosis, CHD, heart fail-
ure, hypertension, PAH, MIRI and atrial fibrillation. However, the impact
of apelin/APJ on atherosclerosis and hypertension is still controversial,
and thus the contribution of the molecular pathway to disease protec-
tion in humans remains to be further explored. Although some synthet-
ic agonists of APJ have therapeutic potential for reducing the risk of
cardiovascular diseases [94–96], the safety and long term effect of
these agonists needs to be more fully characterized and better defined.
In addition to these, there are still many problems left to be solved. How
are apelin and APJ regulated in vivo? Are there any other components
required for their function? Are there efficient approaches to enhance
their cardiovascular physiological actions besides APJ agonists? What
is the exact diagnostic and prognostic role of apelin/APJ in cardiovascu-
lar disease patients? In answering these and many other questions, we
will assuredly strengthen our chances of developing apelin/APJ-based
therapies to help further decrease cardiovascular disease risk in future
generations.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgment
The authors gratefully acknowledge the financial support from the
National Natural Sciences Foundation of China (81070220 and
81170278), and the Aid Program for Science and Technology Innova-
tive Research Team in Higher Educational Institutions of Human
Province, China (2008-244).
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