Current Opinion in Investigational Drugs 2010 11(3):273-282

© Thomson Reuters (Scientific) Ltd ISSN 2040-3429

REVIEW

Apelin and ACE2 in cardiovascular disease

Anastasia Z Kalea & Daniel Batlle*
Address
Northwestern University, Feinberg School of Medicine, Division of Nephrology/Hypertension,
303 East Chicago Avenue, Tarry Building 4-750, Chicago, IL 60611-3008, USA
Email: d-batlle@northwestern.edu

*To whom correspondence should be addressed

Apelin is a peptide that has been identified as the endogenous ligand for the receptor APJ. The apelin/APJ system may be an important
factor in the regulation of vascular tone and cardiovascular function. Studies on cultured cells and small animal models have revealed
that apelin and APJ are localized in cardiomyocytes and vascular cells. The infusion of apelin affects vascular tone and blood pressure,
with both central and peripheral actions. In clinical conditions such as heart failure and atherosclerosis, the gene expression of APJ and
apelin, as well as the levels of circulating apelin, may be altered. The only known active homolog of ACE, ACE2, hydrolyzes apelin with
similar potency to angiotensin II and, therefore, is responsible for the degradation of both peptides. Emerging data on a potential
interaction between the two pathways suggest that the function of apelin/APJ in the vasculature may be relevant to cardiovascular disease,
and identifying how this system is regulated could be useful clinically.

Keywords ACE2, apelin, APJ, cardiovascular disease, endothelium, receptor, vascular tone

Introduction acts as a coreceptor for HIV-1 strains [17] and shares

An increasing amount of scientific evidence during the
past decade suggests a role for the peptide apelin and its
receptor APJ in the cardiovascular system. A combination
of in vitro cellular studies, rodent models of disease, and
data from several human studies has demonstrated that
the apelinergic system is a critical factor in vasomotor
tone [1,2], cardiac contractility [3] and blood pressure
[4-6], and has also been associated with the development
and progression of vascular pathologies [7-10]. Although
information on the metabolism of apelin and APJ is
limited, at the catabolic level, ACE2 is known to cleave
apelin-36 and hydrolyze apelin-13, rendering these
proteins inactive [11]. Several reviews have presented
data on the general role of the apelinergic system in
mammalian physiology [12-14]. This review presents an
overview of the apelinergic system in the vasculature,
highlighting data from the past few years. The
physiological relevance of the degradation of apelin
isoforms by ACE2 is also discussed.
The ligand apelin and its receptor APJ
Apelin, the endogenous ligand for the APJ receptor, was
first extracted from bovine stomachs [15]. The gene
encoding APJ is located on band q12 of chromosome 11,
and codes for a protein of 377 amino acids and 50 to
60 kDa, depending on its glycosylation state. APJ is an
N-glycosylated GPCR consisting of seven transmembrane
domains, and includes a signal sequence in the third
intracellular loop that allows agonist-independent nuclear
localization, a feature that may be cell-specific [16]. APJ
significant homology with angiotensin II (AngII) receptor
type 1 (AT1), with 115 (30%) of the total APJ receptor
structure and 86 amino acids (54%) of the hydrophobic
transmembrane regions being identical with those of
AT1 [18]. However, apelin does not bind to AT1, and AngII
does not bind to APJ. The apelin gene, located on band
q25 to 26.1 of the X chromosome, encodes an initially
translated 77-amino acid pre-proprotein, which is cleaved
by proteases to shorter forms (Figure 1). The 41 N-terminal
amino acids represent secretory signal sequences, while
the remaining 36 C-terminal amino acids comprise
apelin-36. Preproapelin exists as a dimeric protein in vivo
in native tissues, while apelin-36 and apelin-13 are
monomeric [19]. Apelin immunoreactivity was detected
as a band of approximately 16 kDa in different tissue
lysates [19]. Apelin-36, along with smaller isoforms (eg,
apelin-19, -17, -16, -13 and -12) bind to APJ; apelin-36 and
apelin-13 have been detected in different tissues in vivo
[20]. The biological importance of the apelin peptide
is suggested by its wide tissue distribution and by the
strict conservation of the last 13 amino acids at the
C-terminal (amino acids 65 to 77) among all species
studied [5,15,21].
Tissue concentrations of apelin are higher than in plasma
(concentrations in the plasma are in the picomolar
range, as is common for circulating hormones). The sources
of the pool of different apelin isoforms have not been
determined. Apelin is produced by adipocytes [22] and,
in the heart, the atria have been suggested as a site
of production [7]. In general, the biological activity of
274 Current Opinion in Investigational Drugs 2010 Vol 11 No 3
Figure 1. The biologically active isoforms of apelin.
Preproapelin H2N
Apelin-36
Apelin-17
Apelin-13
[Pyr1]apelin-13
The apelin gene encodes an initially translated 77-amino acid pre-proprotein that is cleaved by proteases to form shorter biologically active
forms, such as apelin-36, apelin-17 and apelin-13, as well as the pyroglutamate form of apelin-13 ([Pyr1]apelin-13), which has been used in
several in vitro and in vivo studies [2,19,20,23] and which is resistant to degradation.
(Adapted with permission from Anastasia Z Kalea and Daniel Batlle. © 2010 Anastasia Z Kalea and Daniel Batlle)
apelin peptides is inversely correlated with peptide length.
Kawamata et al combined gel filtration and enzyme
immunoassay data to describe the different tissue
distributions of the long and short apelin isoforms in rats
(ie, sizes close to apelin-36 and apelin-13, respectively);
longer apelin isoforms were expressed in the lung, testis
and uterus, and both the long and short apelin isoforms
were observed in the mammary gland [20]. The
pyroglutamate form of apelin-13 ([Pyr1]apelin-13), which
has been used in various in vitro and in vivo studies,
appears to be resistant to degradation [2]. In several
in vitro studies, apelin-12 was more active in lowering
blood pressure than apelin-13 and apelin-36 [4]. In
addition, a recent study suggested that APJ activation
by [Pyr1]apelin-13, apelin-13 and apelin-36 in the
cardiovascular system occurred with comparable potencies
[23]. The 12 amino acids at the C-terminal form the
shortest active apelin sequence; apelin-11 and shorter
peptides are inactive [4]. Modifications of the terminal
residues in apelin peptides demonstrated that the last
C-terminal phenylalanine is a critical factor in the peptide
structure, and was important to enable apelin to maintain
its blood pressure-lowering effects in Wistar-Kyoto
normotensive (WKY) and spontaneously hypertensive
(SHR) rats [19].
Apelin and APJ are expressed in the brain (hypothalamus),
stomach, gastrointestinal tract, heart, kidney, adipose
tissue, lung and endothelial cells [24-27]. The rat homolog
of APJ, B78/Apj, was detected in the lung, heart, skeletal
muscle, kidney, brain, liver, ovary and anterior pituitary
[28]. In the cardiovascular system, both apelin and
APJ appear to be present in cardiac myocytes and
vascular smooth muscle cells (VSMCs) [25,29], while
immunoreactive apelin has been detected in endothelial
cells [14,25]. APJ immunoreactivity was detected in
endothelial cells lining intramyocardial, renal, pulmonary
and adrenal vessels, as well as in endocardial endothelial
cells [24]. Katugampola et al documented apelin binding
1 to 77 COOH
H2N 42 to 77 COOH
H2 N 61 to 77 COOH
H2N 65 to 77 COOH
H2N-PyroGlu 65 to 77 COOH
sites in the human heart and saphenous vein [2]. In mice,
APJ is expressed in the endothelium of the primary
vessels during embryonic development and in retinal
cells during postnatal development [30]. Ashley et al
demonstrated that, during developmental stages
(ie, embryonic heart development in mice), apelin and
APJ mRNA were coexpressed in endothelial and
myocardial cells, suggesting an autocrine role for this
pathway [31]. After late gestation and in adult life, apelin
was expressed primarily by endothelial cells and APJ
was present in the myocardium, implying that apelin
affects APJ via a paracrine signaling pathway. Using an
apelin+/LacZ reporter mouse, Sheikh et al demonstrated
that the endothelium was responsible for the production
of apelin, even in compromised conditions [32]. The
expression patterns of APJ and apelin in the adult
vasculature remain to be characterized in healthy and
diseased vessels.
The regulation of apelin by ACE2
Limited information is available on the synthesis of
biologically active apelins from immature preproapelin and
on the catabolism of apelin peptides to less active forms.
Apelin-36 and apelin-13 are the circulating peptides that
can currently be detected in plasma using commercial
assays. The only enzyme known to regulate apelin
metabolism is ACE2, a zinc-containing carboxymono-
peptidase that hydrolyzes AngII to produce Ang(1-7),
a vasodilatory peptide. ACE2 also acts on AngI to
produce Ang(1-9), a peptide that is not biologically active
[33]. Using liquid chromatography-mass spectrometry
detection, Vickers et al reported that ACE2 hydrolyzed
apelin-13 with high catalytic efficiency and also cleaved
apelin-36, rendering the proteins inactive (Figure 2) [11].
The hydrolysis of apelin by ACE2 involves the cleavage
of the terminal amino acid, phenylalanine; the hydrolysis
has comparable kinetics with AngII degradation [11].
AngI hydrolysis by ACE2 is less efficient and has a lower
turnover number [11,33]. Other peptides cleaved by

Figure 2. ACE2 and apelin hydrolysis.
Apelin-36
H2N 42 to 77 COOH
ACE2
H2N 42 to 76 COOH + Phe
ACE2 can cleave apelin isoforms by removing the last phenylalanine amino acid from the C-terminal of apelin-36 and apelin-13.
(Adapted with permission from Anastasia Z Kalea and Daniel Batlle. © 2010 Anastasia Z Kalea and Daniel Batlle)
ACE2 with substantial catalytic efficiency include the
opioid peptide dynorphin A1-13, des-Arg9-bradykinin,
β-casomorphin and neocasomorphin [11].
In terms of the distribution of ACE2 in mammalian
tissues, APJ and AT1, as well as apelin and AngII, share
similarities in their tissue-expression patterns. Tissues
with high levels of ACE2 include the kidney, testes,
intestine and heart [34,35]. ACE2 has also been observed
in the endothelial cells of intramyocardial and coronary
blood vessels and in the smooth muscle of small renal
vessels [36-39]. In the kidneys, ACE2 is localized in
glomerular epithelial cells (podocytes) and within tubular
structures, where it is abundantly expressed [39].
Apelin/APJ cellular signaling
Apelin stimulates APJ via the α-subunit of a pertussis
toxin-sensitive Gi2-protein in a Ras-independent, but
PKC-dependent, pathway [40] (Figure 3). Apelin/APJ
binding and APJ activation transduce signals via MAPKs
from the cell membrane level to nuclear targets. Apelin-36
and apelin-13 regulate the same effectors by inhibiting
adenyl cyclase in vitro (apelin-36 was observed to be
less efficient than apelin-13); by signaling through
Gi1/Gi2-proteins, phosphorylation of ERK1/2 and Akt
(Ser473) occurred in CHO and HUVEC cells [41,42].
Bai et al confirmed this signaling pathway for apelin-13
in human embryonic kidney cells (HEK293) [43].
Interestingly, these apelin isoforms differed in their APJ
receptor-desensitization patterns; apelin-36 promoted
a persistent activation of APJ, while apelin-13 activated
APJ transiently and rapidly, given that APJ was
internalized and recycled to the plasma membrane
within 1 h. Longer apelin isoforms lock some receptors
in the active state, exhibit a low dissociation rate from
the receptor and extend the duration of APJ activation
[26,42]. The majority of the studies described were
conducted in vitro in transfected or adeno-infected cells
expressing APJ.
Apelin/APJ and cardiovascular physiology
Early studies reported a hypotensive role of apelin when
the protein was administered intravenously in rats
[4-6]. Apelin was later discovered to have a biphasic
Apelin and ACE2 in cardiovascular disease Kalea & Batlle 275
Apelin-13
H2N 65 to 77 COOH
ACE2
H2N 65 to 76 COOH + Phe
role in vasomotor tone, consisting of two components:
endothelium-dependent and endothelium-independent
actions (Figure 4). Apelin-13 binding to APJ in vascular
endothelial cells activates endothelial nitric oxide (NO)
synthase (eNOS) by phosphorylating Akt. The NO that
is produced diffuses from the endothelial cell to the
VSMCs to induce vasodilation [4,41]. Tatemoto et al
evaluated the administration of apelin-36, apelin-13 and
apelin-12 to anesthetized WKY rats. All apelin isoforms
significantly lowered arterial blood pressure in a dose-
dependent manner, with no effect on heart rate [4].
Apelin-12 was more potent than apelin-13 and apelin-36,
reducing arterial pressure in an eNOS-dependent
manner that was accompanied by increased plasma
concentrations of nitrite/nitrate [4]. The effect of apelin
on the increased l-arginine uptake/eNOS/NO-mediated
effect on vasodilation was confirmed ex vivo in WKY aortae
[44], in AngII-preconstricted juxtamedullary afferent and
muscular efferent arterioles [45], and in isolated aortic
rings and renal artery preparations of spontaneously
diabetic (db/db) adult mice [1,46]. The ability of apelin to
attenuate AngII-induced vasoconstriction in db/db mice
appears to involve the PI3K/Akt-eNOS signaling pathway,
and is abolished in the presence of an AT1-receptor blocker
[1,46]. In conscious Sprague-Dawley rats, apelin-12 was
demonstrated to be both an arterial and venous dilator
in vivo. The depressor effect of apelin was accompanied
by increased heart rate when infused in conscious
animals, possibly because of the hypotension-induced
reflex activation of the sympathetic nervous system,
and was abolished by ganglion blockade [6].
In human saphenous veins disrupted of the endothelial
layer, [Pyr1]apelin-13 caused concentration-dependent
contraction, with high potency but low efficacy [2].
In vivo experiments on human dorsal veins of the hand
revealed that apelin-36 and [Pyr1]apelin-13 had no
effect on vascular tone, but both apelin isoforms caused
reproducible NO-dependent vasodilation in forearm
resistance vessels [47]. Indeed, apelin regulated
vasoconstriction via APJ binding by inducing the
phosphorylation of the myosin light chain (in isolated rat
VSMCs in culture and freshly isolated mouse thoracic
aortae) in a dose-dependent manner via PKC activation
[29], and by inducing a slight increase in intracellular
276 Current Opinion in Investigational Drugs 2010 Vol 11 No 3
Figure 3. The intracellular signal transduction pathways of apelin/APJ binding.
APJ
Apelin
Gi
α [Ca+2]
β PI3K
γ DAG
Angiogenesis mTOR
eNOS
Vascular
homeostasis
The binding of apelin to APJ at the cell surface leads to the rapid transduction of intracellular signals via MAPKs involving ERK and
Akt phosphorylation.
DAG Diacylglycerol, eNOS endothelial nitric oxide synthase, Gi inhibitory G-protein, IP3 inositol triphosphate, PLC phospholipase C
(Adapted with permission from Anastasia Z Kalea and Daniel Batlle. © 2010 Anastasia Z Kalea and Daniel Batlle)
Ca2+ concentrations in renal medullary arterioles in the
absence of a functional endothelium [45].
Although apelin binding to APJ in peripheral tissues
induces vasodilation via a NO-mediated pathway, in
the myocardium, apelin enhances inotropy. Apelin-16
increased mean arterial filling pressure and cardiac
contractility in the isolated perfused rat heart through
the stimulation of Ca2+ influx and an increase in
phospholipase C and PKC, and also increased intracellular
Ca2+ via an increase in Na+/H+ exchange and reverse-
mode Na+/Ca2+ exchange [3]. Berry et al reported the
ex vivo positive inotropic effects of apelin in healthy WKY
rat hearts with a continuous intravenous infusion of the
peptide [48]. Ashley et al investigated the acute and
chronic (using implanted minipumps for 2 weeks) effects
of [Pyr1]apelin-13 in C57Bl/6 mice [31]. Apelin reduced
left ventricular preload and afterload, and enhanced
contractile reserve through an increase in elastance,
without causing hypertrophy. In a study by Kagiyama
et al, both the central and peripheral injection of
[Pyr1]apelin-13 in conscious WKY rats increased mean
arterial pressure and heart rate – effects that were
abolished by AT1 receptor blockade [49]. This finding
on the central action of apelin is supported by the more
recent observation that the acute microinjection of
apelin-13 or viral vector-mediated apelin gene transfer
into the rostral ventrolateral medulla (the site of
apelin overexpression in SHRs) induced the chronic
elevation of blood pressure [50].
The observed interruptions of apelin/APJ effects in the
presence of an AT1 receptor blocker [1,46,49] suggested
PLC IP3
PKC
ERK
Akt
Transcription
Nucleus
Cytoskeleton
a signaling interaction with the AngII/AT1 signaling
pathway. The physiological relevance of this interaction
was investigated by Gurzu et al, who reported that
the systemic administration of apelin-13 alone did not
modify the vascular tone of isolated WKY rat portal vein
specimens but, rather, suppressed the AngII- and
phenylephrine-induced contractions in the presence of
endothelium in an eNOS/inducible NOS-mediated manner,
and also (to a smaller extent) when the endothelium
was disrupted [51]. The intracerebroventricular
administration of apelin-13 in conscious mice directly
inhibited vasopressin release via APJ in vasopressinergic
neurons [52]. This observed effect contrasts the effect
of AngII, which stimulates vasopressin release by
stimulating the AT1 receptor [53]. Reaux et al reported
that apelin colocalized with vasopressinergic neurons in the
supraoptic nucleus, and that the intracerebroventricular
administration of apelin-13 in conscious mice with free
water access inhibited vasopressin release via APJ
binding [54]. This counteracting role of apelin on
vasopressin was also confirmed by later studies [55].
To further elucidate the biological role of apelin and APJ,
two separate research groups attempted to generate
apelin knockout and APJ knockout mouse strains. When
Ishida et al generated APJ-/- mice and double-knockout
mice for APJ and AT1a, a similar baseline systolic blood
pressure and heart rate was observed between APJ-/-
and wild-type mice, suggesting that, under baseline
conditions, APJ is not essential for the maintenance of
blood pressure [56]. The systemic (intraperitoneal)
administration of [Pyr1]apelin-13 in conscious male mice
lowered systolic blood pressure, but not heart rate,
 Apelin and ACE2 in cardiovascular disease Kalea & Batlle 277

Figure 4. Apelin and vasomotor tone in isolated vessels.

Apelin APJ

IP3 Endothelial
Gi Gq PLC cell
α
α [Ca+2] β
β P13K γ
γ DAG
CaM
L-Arginine
PKC Akt eNOS
ERK
L-Citrulline
+
Transcription NO
Nucleus

Vascular smooth
IP3 + DAG muscle cell
Gq PLC NO
α [Ca+2] PKC
β PI3K
γ
PIP2 CaM sGC
cGMP GTP cAMP

MLC MLCK Pi –
[Ca+2]
Phosphatase + +

Vasoconstriction Vasodilation

The vasomotor intracellular effects of apelin/APJ interaction in endothelial and vascular smooth muscle cells (VSMCs) are shown.
Apelin isoforms bind to APJ in endothelial cells and in VSMCs, and affect vasodilation and vasoconstriction signaling pathways.
CaM Calmodulin, cGMP cyclic guanosine monophosphate, DAG diacylglycerol, eNOS endothelial nitric oxide synthase, ER endoplasmic
reticulum, Gi inhibitory G protein, Gq phospholipase C-activating G protein, GTP guanosine triphosphate, IP3 inositol triphosphate,
MLC myosin light chain, MLCK MLC kinase, NO nitric oxide, PIP2 phosphatidylinositol 4,5-bisphosphate, PLC phospholipase C, sGC soluble
guanylyl cyclase
(Adapted with permission from Anastasia Z Kalea and Daniel Batlle. © 2010 Anastasia Z Kalea and Daniel Batlle)

only in the presence of APJ and in an eNOS-dependent
manner. When AngII was administered at low doses,
APJ-/- mice were more sensitive to the hypertensive
effects of the protein compared with wild-type mice [56].
In a study by Kuba et al, apelin gene-targeted mice
developed heart failure in response to pressure overload,
but exhibited a normal heart structure, similar APJ mRNA
levels and, contrary to expectations, the same blood
pressure as the wild-type mice, even when AngII was
infused. The slight decrease in blood pressure that was
observed in conscious aged apelin-/- mice was attributed
to their impaired cardiac contractility. The infusion of
apelin-13 in these animals reversed the decreased
contractility resulting from aging [57]. Given that ACE2
hydrolyzes and inactivates apelin-13, ACE2-/- mice might
be expected to exhibit an opposite phenotype compared
with apelin-/- mice. However, evidence from studies on
the ACE2-/- mouse models supports the observation that
these mice develop abnormal heart function and impaired
cardiac contractility, as similarly occurs in aged, male
animals.
Both apelin and APJ are overexpressed during
developmental phases in the vasculature [58] and in the
developing retina [30], implying a role for the proteins in
vessel formation. Cellular functions, such as endothelial
cell migration, proliferation and the formation of
capillary tubes, are crucial steps during angiogenesis.
Kidoya et al demonstrated that apelin induced endothelial
cell aggregation and proliferation in a VEGF-dependent
278 Current Opinion in Investigational Drugs 2010 Vol 11 No 3
manner, affecting the formation of larger-diameter
vessels during angiogenesis [59]. The in vitro observed
apelin-dependent stimulation of HUVEC migration,
proliferation and in vitro tube formation (which is blocked
when APJ is silenced) was also demonstrated in vivo
in a murine model of adipose tissue-derived
angiogenesis [60] and in an embryonic and tumor model
of angiogenesis [61].
Apelin/APJ in cardiovascular pathologies
Following the availability of suitable biochemical
measurement kits, recent studies have reported
associations between circulating apelin levels and
cardiovascular disease. The variability observed among
these studies may be attributed to differences in the
populations tested, but also because of unknown factors
involved either in apelin synthesis and maturation or in
apelin proteolysis and degradation. The distinctly different
commercial methods available for the measurement of
circulating apelin levels in plasma may also contribute to
this variability.
The potential of apelin/APJ to reduce cardiac preload and
afterload and to increase contractile reserve, with no
effect on cardiac hypertrophy, has increased interest in
the role of this system in heart failure. The observations
from studies in both rodents and humans suggest that
prolonged cardiac afterload induces the upregulation of
apelin and APJ gene expression as a compensatory and
adaptive mechanism, and this upregulated expression
is subsequently downregulated during later stages of
heart failure [3,62,63]. The placement of a left ventricular
assist device [63] or the use of an AT1 receptor blocker
[62] in failing hearts restored apelin and APJ cardiac
expression. Plasma apelin levels also appeared to be
decreased in end-stage heart failure and in myocardial
infarction [7,64,65]. Using a radioimmunoassay specific
for apelin-36, Földes et al demonstrated decreased
circulating levels of apelin-36 in patients with heart
failure, although apelin mRNA levels were similar in the
atria of failing and healthy hearts [7]. Apelin-13 infusion
in a rat model of ischemic cardiomyopathy resulted in
the improvement of cardiac function in both sham and
failing hearts [8]. With a novel ex vivo hemodynamic
monitoring system, Berry et al confirmed that the
inotropic effects of apelin-16 were maintained in ischemic
cardiopathic hearts in rats [48], while genetic studies
using apelin+/LacZ mice confirmed the observations that
apelin/APJ is upregulated in an early compensated phase of
heart failure and is linearly correlated with disease stage
and severity [32]. In the failing human heart, ACE2 gene
expression is upregulated [66,67]. The blockade of AT1
receptors (by losartan or olmesartan) increased cardiac
ACE2 mRNA expression and activity in the ischemic
myocardium [68,69]. Studies on apelin hydrolysis by
ACE2 in vivo are lacking, however, and such observations
can therefore only be assumed to have physiological
relevance. In progressing cardiomyopathy, the observed
upregulation of ACE2, evaluated mostly by an increase
in Ang(1-7), has also been proposed to facilitate the
breakdown of biologically active apelins that are
concurrently transcriptionally downregulated [62,70]. Using
an animal model of MCT-induced pulmonary hypertension,
Falcao-Pires et al reported that [Pyr1]apelin-13 infusion
decreased right ventricular overload and hypertrophy, but
induced endogenous apelin and Mas receptor myocardial
expression both in healthy and diseased animals; in
animals with pulmonary hypertension, [Pyr1]apelin-13
increased APJ and decreased AngII and endothelin 1
(ET1) gene expression, while preventing angiotensinogen
formation [70]. In pulmonary hypertension, circulating
levels of plasma apelin-36 were reported to be decreased
3-fold in patients with chronic parenchymal lung disease
but normal cardiac function, when compared with
patients with chronic heart failure, suggesting that
plasma levels of apelin may be a diagnostic tool for
patients with severe pulmonary disease [71]. However, in
an animal model of pulmonary hypertension, Andersen
et al recently reported that, while apelin protein
concentration in lung tissue decreased, the total
pulmonary apelin content remained stable because of
a corresponding increase in pulmonary tissue mass;
in addition, in pulmonary arteries from normoxic rats,
apelin counteracted vasoconstriction in response to ET1
that was abolished in hypoxic rats [72].
Based on the role of apelin/APJ in the vasculature, several
researchers have attempted to determine the role of
these proteins in conditions involving endothelial
dysfunction, such as atherosclerosis. APJ-/-/ApoE-/- double-
knockout mice fed on a high-cholesterol diet demonstrated
a reduced atherosclerotic lesion size when compared
with APJ+/+/ApoE-/- mice and exhibited decreased vascular
oxidative stress (as measured by the expression of
NADPH oxidase subunits and the production of superoxide
radicals) while on a healthy diet [73]. The presence of
[Pyr1]apelin-13 in extracted VSMCs in vitro stimulated
the mRNA expression of NADPH oxidase subunits and
increased APJ-dependent VSMC proliferation; the VSMC
proliferation was reversed by superoxide dismutase
[73]. Although these findings support a role for APJ in
hypercholesterolemia-associated atherosclerosis, a study
on apelin-/-/ApoE-/- double-knockout mice demonstrated
that apelin antagonized a series of AngII-related
atherosclerotic effects [10]. Apelin infusion in ApoE-/- mice
increased NO bioavailability, decreased atherosclerotic
lesion size and aortic superoxide levels, and inhibited the
AngII-mediated increase in neointimal formation [10].
The closely associated receptors APJ and AT1 appear to
cross-talk and, interestingly, APJ inhibited AngII/AT1
signaling independent of apelin [10]. In a group of
well-characterized healthy individuals with isolated
dyslipidemia, plasma apelin was decreased and
correlated only with HDL levels; however, plasma apelin
was increased after treatment for the reduction of
LDL-cholesterol levels, both with therapeutic lifestyle
change or statin treatment [74]. In patients with stable
angina, plasma apelin was negatively correlated with
the extent of coronary stenosis [75]. The increased
systemic oxidative stress in patients with stable angina,

demonstrated by increased plasma levels of 8-iso-PGF2α,
as well as the correlation of plasma apelin with
endothelial cell injury markers [76], support the
hypothesis that apelin-APJ affects lipid peroxidation and,
therefore, atherosclerosis. Both early and advanced
carotid atherosclerotic lesions in humans also express
ACE2 mRNA, with ACE2 total vessel expression being
independent of the stage of atherosclerosis. However,
ACE2 activity was reported to be decreased in advanced
atherosclerotic lesions, compared with early and ruptured
atherosclerotic lesions [77]. Lovren et al demonstrated
that ACE2 was important for endothelium-dependent
relaxation, endothelial cell migration and tube formation,
and for the inhibition of monocyte and adhesion
molecule expression [78]. ACE2 partially attenuated the
AngII-induced production of reactive oxygen species
and improved endothelial homeostasis in an Ang(1-7)-
dependent manner. More mechanistic studies are
necessary to confirm the effect of ACE2 on the apelin-APJ
system in the pathology of atherosclerosis and in other
inflammation-related pathologies, such as diabetes.
Hypoxia is a major factor regulating apelin/APJ gene
expression and/or secretion in cardiac myocytes [32,79].
Apelin, which is predominantly expressed in endothelial
cells, is induced by hypoxia via the hypoxia-inducible factor
(HIF)-1α (ubiquitous) and HIF-2α (endothelium-specific
expression) pathways [32,79]. In the hearts of ACE2-/-
mice, hypoxia-inducible genes (BNIP3 and PAI-1) are
upregulated [80,81] and the localized AngII levels in the
heart are increased [36], with no signs of cardiac
hypertrophy. Whether hypoxia alters ACE2 expression
levels remains to be demonstrated. Apelin gene expression
is downregulated in the presence of AngII (in cardiac
cells) [62], growth hormone and TNFα (mouse 3T3-L1
adipocytes) [82,83], by shear stress in HUVECs [59], in
cultured rat ventricular myocytes subjected to mechanical
stretch [3], in the left ventricles of two experimental
models of chronic pressure overload in vivo [3] and in
models of essential hypertension [84]. However, in
many cases, whether the changes in mRNA expression
correspond to an effect in post-translational protein
synthesis and activity remains unknown.
Several novel, cyclin apelin analogs have been developed
from the minimal C-terminal 12-amino acid apelin
fragment to facilitate the study of apelin bioactivity [85].
Some studies have also used synthetic apelin fragments
to demonstrate that the last two amino acids from the
apelin C-terminus do not eliminate its APJ-binding ability,
nor its ability to inhibit cAMP production in vitro;
however, in a study in which the apelin fragments
were injected into WKY mice, the peptides were not
sufficiently efficient to reduce mean arterial blood
pressure or to induce an increase in heart rate when
compared with the full 17-amino acid fragments because
of their inability to induce APJ internalization and recycling
[86]. Recently, Iturrioz et al identified and synthesized
the first non-peptidic agonist for the apelin receptor,
E339-3D6, which functions as a partial agonist of cAMP
Apelin and ACE2 in cardiovascular disease Kalea & Batlle 279
production and as a full agonist of apelin receptor
internalization [87]. E339-3D6 induced the vasorelaxation
of rat aorta precontracted with norepinephrine, and
potently inhibited systemic vasopressin release in
water-deprived mice when administered intracerebro-
ventricularly; this apelin agonist also decreased
vasopressin release centrally [87].
Conclusion
Peptides of the apelin family are hydrolyzed by ACE2,
which renders them inactive; this hydrolysis has
comparable kinetics to the hydrolysis of AngII by ACE2.
The newly identified roles of the apelinergic system
in cardiovascular function include an endothelium-
mediated NO-dependent hypotensive effect of small
apelin isoforms in large vessels that requires APJ
binding and a vasoconstricting effect in VSMCs when the
endothelium is disrupted, and a positive inotropic effect
in the myocardium, increasing heart contractility. Overall,
the apelinergic system appears to regulate AngII
signaling in the vascular system, although the effects of
apelin administration in pathological animal models has
required a high dose of the peptide [88]. The regulation
of AngII/AT1 signaling by the endogenous apelin/APJ
pathway in the vasculature warrants further study;
interestingly, ACE2, an important enzyme in the apelin/
APJ pathway, is the only known metabolic mechanism
that decreases apelin bioavailability. Assuming a
vasoregulatory role of apelin in the presence of a
functional endothelium, future studies should elucidate
the role of ACE2 in maintaining the balance of apelins in
both healthy and diseased conditions. Unraveling the role
of ACE2, apelin/APJ and AngII/AT1 in the cardiovascular
system may help to identify key targets for therapeutic
intervention.
Acknowledgements
Anastasia Z Kalea is a recipient of a postdoctoral
fellowship from the Juvenile Diabetes Research
Foundation International. Daniel Batlle has received
support from the NIH (R01 DK080089) and the Juvenile
Diabetes Research Foundation International (7-05-RA-06).
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Apelin-13 and apelin-36 exhibit direct cardioprotective
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