Apelinergic system in endothelial cells and its role in angiogenesis in myocar-
dial ischemia

Vera Novakova, Gurpreet S. Sandhu, Dan Dragomir-Daescu, Martin

Klabusay

PII: S1537-1891(15)00181-0
DOI: doi: 10.1016/j.vph.2015.08.005
Reference: VPH 6244

To appear in: Vascular Pharmacology

Received date: 16 April 2015

Revised date: 1 August 2015
Accepted date: 3 August 2015

Please cite this article as: Novakova, Vera, Sandhu, Gurpreet S., Dragomir-Daescu, Dan,
Klabusay, Martin, Apelinergic system in endothelial cells and its role in angiogenesis in
myocardial ischemia, Vascular Pharmacology (2015), doi: 10.1016/j.vph.2015.08.005

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 ACCEPTED MANUSCRIPT

APELINERGIC SYSTEM IN ENDOTHELIAL CELLS AND ITS ROLE IN ANGIOGENESIS IN

MYOCARDIAL ISCHEMIA

1
Vera Novakova, 2,4Gurpreet S. Sandhu, 3,4Dan Dragomir-Daescu, 5,6Martin

T
Klabusay

P
RI
1 Integrated Center of Cellular Therapy and Regenerative Medicine, International Clinical Research
Center, St. Anne’s University Hospital, Brno, Czech Republic

SC
2 Division of Cardiovascular Diseases, Mayo Clinic, Rochester, MN 55905 USA

3 Division of Engineering, Mayo Clinic, Rochester, MN 55905 USA

NU
4 College of Medicine, Mayo Clinic, Rochester, MN 55905 USA

5 Department of Internal Medicine - Cardiology, Palacky University, Olomouc, Czech Republic

MA
6 Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech
Republic

Corresponding author at: Martin Klabusay, Regional Centre for Applied Molecular Oncology,
Masaryk Memorial Cancer Institute, Zluty kopec 7, Brno 656 53, Czech Republic;
ED

Tel.: +420 603 167 625

E-mail address: m.klabusay@sky.cz
PT

Conflict of interest: The authors declare no conflicts of interest.

CE

Abstract
AC

Apelin is a peptide known to have a vital role in cardiovascular diseases. It has been proven to induce
proliferation and tube formation in endothelial cells, stabilise contacts between endothelial cells, and
mediate pericyte recruitment. Since apelin level is reduced early after myocardial infarction, a
supportive therapy with apelin is being investigated for its beneficial effect on blood vessel
formation. It is becoming apparent, however, that the final effect of apelin often depends on stimuli
the cell receives and the cross-talk with other molecules inside the cell. Hence, understanding the
apelin pathway potentially can help us to improve angiogenic therapy. This review summarises
recent knowledge regarding molecules involved in apelin signalling while focusing on their roles in
angiogenesis within the ischemic environment after myocardial infarction.

Keywords:

apelin, angiogenesis, endothelial cells, myocardial infarction, signalling pathways

Chemical compounds

apelin-13 (PubChem CID: 71433878); [Pyr1]-apelin-13 (PubChem CID: 25078060); apelin-12

(PubChem CID: 479167); apelin-36 (PubChem CID: 16130451)
 ACCEPTED MANUSCRIPT

1. Introduction

Angiogenesis is impaired in many diseases, such as myocardial infarction (MI), atherosclerosis and
diabetes. Understanding the pathways of angiogenesis helps us to find new therapeutics or improve
already established methods for restoring blood flow. Since the first angiogenic factors were purified

T
in the 1980s, many new factors connected to angiogenic pathways have been discovered. To name

P
just a few, these include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF),
and angiopoietin-1 (Ang-1) [1-3].

RI
In relation to acute MI, pro-angiogenic therapy has emerged as having promising potential in cardiac

SC
repair [4-6]. Nevertheless, recovery of cardiac function in acute myocardial infarction (AMI) patients
is not always satisfactory [7]. It appears that delivery of a single growth factor or cell type is not
sufficient for long-term cardiac repair. New strategies are therefore being investigated, including
co-delivery of endothelial progenitor cells and vascular smooth muscle cells (SMCs) [8],

NU
pre-conditioning of transplanted cells [9], and application of genetically modified stem cells [10, 11].

A combined gene therapy using several growth factors appears to be more efficient than is delivery
MA
of a single growth factor [12, 13]. This approach is based on the multi-step process of angiogenesis
regulated in vivo by several angiogenic factors [14]. Importantly, overexpression of a single
pro-angiogenic factor may induce pathologic blood vessel formation and even lead to formation of
vascular tumours [15]. Accordingly, emphasis is given to regulating stabilisation and maturation
ED

processes together with preserving a balance of pro-angiogenic and anti-angiogenic factors. New
modifications and regulators of angiogenic therapy are being tested to further improve therapy [16,
17]. One of the angiogenesis regulators intensively studied in recent years is apelin, which has been
PT

found to play an important role in blood vessel formation [17-21]. This review discusses how apelin
and other molecules involved in the apelin pathway influence angiogenesis while focusing on those
molecules’ roles in ischemia after AMI.
CE

2. Apelin isoforms and distribution

Apelin is a peptide first identified in its 36-amino acid isoform in 1998 in bovine stomach extracts
AC

[22]. It binds to a G-protein-coupled angiotensin II protein J (APJ) receptor [22, 23]. Several active
isoforms of apelin have been identified to date. The isoforms are produced by cleavage of 77-amino
acid pre-proapelin [24], bind to APJ receptor, and cause similar cellular effects [25]. However, shorter
peptides consisting of 13 to 17 amino acids have higher activity than does apelin-36 [22, 26]. In
human heart, apelin-13 is the predominant isoform, while in plasma apelin-13 and apelin-17 can be
found [27-29].

Since its first identification, the apelin/APJ pathway has been described to be active in various cells,
tissues and organs (including heart, brain, lung, kidney, endothelium, plasma, and adipose tissue)
[22, 26, 30]. In the vascular system, apelin and APJ receptor are expressed by endothelial and smooth
muscle cells [17, 31, 32].

3. Apelin in endothelial cells of sprouting vessels

Vessel sprouting during angiogenesis is co-ordinated by signals from nearby tissue and the vessels
themselves [33, 34]. However, the responses of individual endothelial cells to the signals differ
according to their positions in the vessels. Tip cells are specialised endothelial cells (ECs), localised at
the leading edge of the vessels, which secrete cytokines, extend filopodia, and lead the migration in
response to VEGF gradient [34]. The lumen of new vessels, by contrast, is formed of stalk cells which
 ACCEPTED MANUSCRIPT

also secrete various factors [34]. Both tip and stalk cells express apelin, whereas APJ receptor is
located on stalk cells only [35]. Surprisingly, APJ is little expressed in unstimulated ECs, but it is
abundantly expressed under hypoxia and after stimulation with VEGF [36-38]. Although under
normal conditions APJ receptor can be found mostly in the cell membrane, the receptor moves into
the cytoplasm after stimulation with apelin [38].

T
4. Angiogenesis in ischemic tissue and apelin signalling

P
Hypoxia and the need for restoration of insufficient blood supply trigger angiogenesis in ischemic
tissue. After VEGF stimulation, endothelial cells differentiate, proliferate, release platelet-derived

RI
growth factor (PDGF-BB), and attract mural cells (pericytes or smooth muscle cells) to newly formed
blood vessels [39]. In the following phase, the pre-existing vessels are stabilised by mural cells

SC
releasing angiopoietin-1 (Ang-1), thereby activating tyrosine-protein kinase receptor Tie-2 on ECs and
consequently stabilising intracellular junctions [40, 41].

Apelin plays an essential role in blood vessel formation and ischemia repair [42, 43]. It has been

NU
described as vital for all the steps of angiogenesis, upregulating both migration and tube formation
[17] and having beneficial effect on stabilisation of contacts between endothelial cells generating
non-leaky blood vessels [18]. Similarly, APJ receptor is necessary also for functional angiogenesis. In
MA
knockout mice that were APJ-/-, it has been observed that the blood vessels did not form
appropriately and vascular smooth muscle cell layers were decreased even though the level of CD31
endothelial cells was preserved [44].
ED

Hypoxia induces expression of APJ receptor on endothelial cells. This process leads to activation of
apelin pathway [45]. In the next step, apelin produced by tip cells binds to APJ receptor on stalk cells
and stimulates downstream signalling pathways in ECs [35].
PT

To date, a rather large number of signalling molecules have been reported to be implicated in the
apelin pathway during angiogenesis, both affecting apelin expression and acting downstream of
apelin/APJ signalling. We discuss here the signalling networks and role of the molecules in ischemic
CE

tissue.

5. Apelin pathway and cross-talking signalling molecules

AC

5.1 Hypoxia-inducible factor

The key step at the beginning of the apelin pathway is hypoxia. Hypoxia is regulated by hypoxia-
inducible factor (HIF-1), which promotes the expression of genes containing hypoxia-responsive
elements (HREs) [46]. In 2006, HREs were found in the apelin promoter, albeit without confirmed
functionality [47]. One year later, it was demonstrated that apelin can be upregulated in response to
hypoxia in cardiac tissue [48]. Finally, in 2008 it was shown that apelin expression in endothelial and
vascular smooth muscle cells is mediated by the binding of HIF-1 to an HRE located within the first
intron of apelin [49]. (Figure 1) Under hypoxia, apelin induces the proliferation of endothelial cells
independently of the VEGF/VEGF-receptor signalling pathway [45].

5.2 Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs), a subgroup of transforming growth factor-β (TGF-β), have
emerged as critical regulators of vascular development [50, 51]. Recently, apelin has been shown to
be strongly downregulated by BMP signalling (BMP4, BMP7, BMP9, BMP10) in endothelial cells [52-
54]. This inhibition occurs via BMPR-2 receptor and the SMAD pathway [52]. It has been
demonstrated that BMPR-2 receptor together with activin receptor-like kinase 1 (ALK-1) activate
 ACCEPTED MANUSCRIPT

SMAD1,5,8 signalling after BMP9 stimulation and that SMAD4 is also vital for the inhibition of apelin
expression [54]. This signalling pathway is connected with Notch signalling since SMAD signalling
induces expression of Notch transcription factors hairy and enhancer of split-1 (HES1), as well as of
hairy/enhancer-of-split related with YRPW motif proteins 1 and 2 (HEY1 and HEY2). It also co-
operates with activated Notch on induction of the genes [54]. Hence, ALK signalling co-operates with
Notch signalling (delta-like ligand 4, DLL4) to inhibit apelin, thereby inducing expression of the

T
vascular guidance receptor unc-5 homolog B (UNC5B) and of vascular endothelial growth factor

P
receptor 1 (VEGFR1) [54]. BMPs also reduce hypoxia-induced apelin expression and endothelial cell
proliferation [52]. In another study, by contrast, BMP-2 was described as regulating expression of

RI
apelin in pulmonary arterial ECs by formation of a transcriptional complex between peroxisome
proliferator-activated receptors (PPARγ) and β-catenin and consequently promoting endothelial cell

SC
survival, proliferation and migration [55]. The PPARγ/β-catenin pathway seems to be SMAD
independent [56]. (Figure 1). These discrepancies in BMP effect on apelin can be attributed to
several factors. The first factor relates to extracellular regulators of BMP, namely BMP endothelial

NU
cell precursor-derived regulator (BMPER), gremlin and tumour necrosis factor (TNF)-stimulated gene
(TSG), which can promote angiogenesis depending on their concentrations [57, 58]. The second
factor consists in interplay of the BMP signalling pathway with other proteins from the cross-talking
signalling pathways. The last but not negligible factor might relate to the cell type or culture
MA
conditions.

5.3 Angiopoietin/tyrosine-protein kinase receptor signalling

ED

Tyrosine-protein kinase receptor Tie-2, a receptor for angiopoietin-1 (Ang-1), plays an important role
in maturation of blood vessels inducing cell adhesion between mural cells and endothelial cells [59].
Ang-1 also is capable of pericyte recruitment [60]. Angiopoietin-2, on the other hand, competes with
Ang-1, destabilises the vessels [61], and reduces pericyte recruitment [60]. In addition to being
PT

produced by mural cells, Ang-1 can be made by hematopoietic stem cells and progenitor cells that
are found in the perivascular region during angiogenesis [62]. Ang-1 binding to its receptor Tie-2 on
CE

endothelial cells induces apelin production [36] (Figure 1). Interestingly, a feedback loop of apelin
stimulating Ang-1 expression has been reported recently in endothelial cells [21] (Figure 2). Ang-1 co-
delivered with apelin leads to high maturation of vessels, but it does not increase capillary growth
[60].
AC

5.4 Monocyte chemoattractant protein-1

Monocyte chemoattractant protein-1 (MCP-1/CCL2) promotes migration and activation of

monocytes and macrophages [63]. MCP-1 affects angiogenesis, too, either by trafficking of
monocytes into a damaged area [64] or by acting directly on ECs [65]. MCP-1 binds to C-C chemokine
receptor type 2 (CCR2) on vascular smooth muscle cells (VSMCs) and pericytes [19]. MCP-1 is known
to be regulated by nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) [66] and
SMAD transcription factors [67]. In mural cells, MCP-1 is implicated in proliferation and its expression
is induced by Ang-1 [68]. Apelin appears also to regulate MCP-1, but there are controversial findings
with regard to apelin’s effect on MCP-1 expression in endothelial cells. In a recent study, silencing of
apelin with siRNA led to upregulated MCP-1 expression in mouse brain endothelioma cells (bEnd.3)
and this was achieved via the suppression of Akt phosphorylation followed by SMAD3
phosphorylation [19]. Similarly, apelin down regulates MCP-1 expression in macrophages [69]. Via
the MCP-1 signalling pathway, apelin also represses vascular smooth muscle cell migration and
pericyte recruitment [19]. Other studies, however, bring different results. In a mouse model study of
laser-induced choroidal neovascularization, apelin had no effect on the level of MCP-1 expression in
either an apelin-knocked-out mouse or in human umbilical vein endothelial cells (HUVECs) [70]. Lu et
 ACCEPTED MANUSCRIPT

al. [71] found apelin to have the opposite effect on MCP-1, stimulating its expression and secretion in
HUVECs via c-Jun NH2-terminal kinase (JNK) and NF-κB. JNK [72, 73] and NF-κB [73] have been
described to stimulate MCP-1 in other cells, too (Figure 2). Hence, further research will be needed to
reconcile these data, as there can be other factors downstream of apelin/APJ signalling which affect
the induction of MCP-1 expression.

T
5.5 Akt/mTOR/p70S6K and ERK signalling pathway

P
Mitogen-activated protein kinases (MAPKs) and the PI3 kinase/Akt pathway, vital for the control of
cell differentiation, also are known to induce angiogenic differentiation in endothelial cells [74].

RI
Stimulation of these pathways has recently been investigated also in relation to therapeutic
angiogenesis [75]. Apelin has been found to be an upstream regulator of the Akt/mammalian target

SC
of rapamycin (mTOR)/p70S6K and extracellular-signal-regulated kinase (ERKs) pathways. In HUVECs
apelin induces cell proliferation after the phosphorylation of p706S kinase via ERK and PI3 kinase
(PI3K) signalling [76], and in stalk cells the mTOR pathway was reported downstream of apelin [35].

NU
In lymphatic endothelial cells, the phosphorylation of Erk1/2 and Akt stimulated by apelin has been
shown to increase endothelial cell migration and tube formation capacity [77]. Hence, p706S seems
to be a common target of both AKT/mTOR and ERK pathways (Figure 2). P706S has been reported in
MA
several other studies to have a central role in endothelial cell proliferation [78, 79], and it has been
demonstrated also to regulate HIF-1α expression in human ECs [80].

5.6 Adenosine monophosphate-activated kinase

ED

Adenosine monophosphate-activated kinase (AMPK) is activated by increased intracellular

concentrations of adenosine monophosphate (AMP) [81], and in endothelial cells its activity is
regulated by hypoxia [82], fluid shear stress, basic fibroblast growth factor [82], adiponectin [83] and
PT

VEGF [82]. Although AMPK is generally known to be involved in metabolic control, in endothelial cells
it has been reported to be implicated in angiogenesis [82]. In HUVECs, AMPK is activated by hypoxia
via phosphorylation at threonine-172 (Thr-172) [82]. A link between AMPK and apelin has recently
CE

been proven in myocardial microvascular endothelial cells, wherein apelin-13 stimulated the
phosphorylation of AMP-activated protein kinase and endothelial nitric oxide synthase (eNOS) at
Thr-172 and serine-1179 (Ser-1179) [17] (Figure 2). A link between AMPK and apelin was reported
also for energy metabolism in adipose tissue [84] and myocardium [85] while involving additional
AC

proteins, such as insulin receptor substrate-1 (IRS-1) [85]. IRS-1 suggests itself potentially to be
involved in the apelin-AMPK pathway in ECs, as it had been described to be expressed in ECs [86] and
has an ability to mediate angiogenic effects [87].

5.7 Myocyte enhancer factor 2

Myocyte enhancer factor 2 (MEF-2) is a transcription factor expressed in endothelial cells that plays a
role in the cardiovascular system and affects EC proliferation and capillary tube formation [88]. MEF-
2C, one of four existing MEF-2 transcripts, was found to have an inhibitory effect on sprouting by
upregulating alpha-2-macroglobulin (A2M) [89]. It is suggested, however, that this inhibition is
induced under normoxic conditions but not under hypoxia [89]. In contrast, MEF-2A activates EC
proliferation and capillary tube formation via Gα13 [88]. As MEF-2 forms a complex with the
transcriptional repressors, histone deacetylases (HDACs), phosphorylation of HDACs disrupts this
interaction and activates MEF-2. One of the potent activators of MEF-2, which phosphorylates HDAC,
is Ca2+/calmodulin-dependent protein kinase (CaMK) [90]. Recently it has been demonstrated that
MEF-2 (MEF-2A, MEF-2C) expression is also regulated by the apelin/APJ pathway [44] since APJ-/-
mice were found to have significantly decreased MEF-2. Apelin-APJ regulates MEF-2 activation via G-
 ACCEPTED MANUSCRIPT

protein Gα13 and via phosphorylation of HDAC4 and HDAC5 and their transport from nucleus to
cytoplasm (Figure 2). Gα13 is involved also in HDAC4/5 phosphorylation [44].

5.8 Krüppel-like factor 2 (KLF2)

The MEF-2 signalling pathway has been found to be connected with KLF-2, a zinc-finger transcription
factor regulating vascular growth and endothelial function [91, 92]. KLF-2 has been reported to be

T
vital for differentiation and angiogenesis of endothelial colony-forming cells [92]. In addition, KLF-2

P
regulates expression of the tight junction protein occludin in ECs, and therefore KLF-2 deficiency
causes endothelial leakage [93]. KLF-2 is regulated by apelin [94, 95] via the AMP-activated protein

RI
kinase/MEF2 pathway [92, 94] (Figure 2). Recently HDAC5 has been reported to repress KLF-2 in
HUVECs by direct binding in the nucleus [96]. Moreover, HDAC5 can also repress MEF-2 [97]. After its

SC
phosphorylation, however, HDAC5 is exported from the nucleus and MEF-2 can activate KLF-2
expression [97].

6. Apelin/APJ expression in pericytes and smooth muscle cells

NU
Pericytes and smooth muscle cells are essential for the maturation of newly formed vessels. VSMCs
express APJ receptor [98] but do not produce apelin [99]. Apelin has been shown to mediate pericyte
MA
recruitment [60]. Furthermore, apelin has been found to stimulate vascular SMC proliferation by two
distinct signalling pathways. The first described pathway acts via Jagged-1/Notch3 and Cyclin D1 and
has been reported to be dependent on apelin-13 concentration [100]. In the other pathway, apelin-
13 stimulates proliferation and migration of VSMCs by early growth response-1 (Egr-1) transcription
ED

factor up regulation [101]. Both pathways can be abolished by ERK inhibitors, thus suggesting their
cross-talk [100, 101]. Apelin also has been found to stimulate migration and proliferation of
neointimal SMCs in mice after carotid artery ligation [102]. Under high glucose level conditions,
PT

interestingly, apelin acts distinctly on pericyte recruitment [19]. Apelin knockdown in ECs in a murine
model of pathological retinal angiogenesis up-regulated MCP-1 expression in stalk cells and resulted
in the release of pericyte recruitment factor from endothelial cells. This MCP-1-mediated pericyte
CE

recruitment was transduced via PI3-AkT and Smad3 pathway [19].

7. The role of apelin in monocytes/macrophages

AC

Myocardial infarction triggers recruitment of monocytes to the damaged heart, where they
differentiate to become macrophages. These cells perform various functions after MI, including
removing damaged extracellular matrix and dying cardiac cells, releasing inflammatory mediators,
and promote angiogenesis by releasing growth factors, cytokines and metalloproteinases [103, 104].
Interestingly, macrophages have been observed to be able to carve out tunnels in the extracellular
matrix mediating angiogenesis [105]. It is generally considered that these antagonistic roles are
performed by two distinct subsets of monocytes which were identified for the first time in 1989
[Passlick et al., 1989]. Most of the monocytes/macrophages are CD14highCD16low/negative. These are
assumed to be active in the first four days after MI, when they release inflammatory cytokines. The
minority subset is CD14lowCD16high [106, 107] and has reparative function, releasing anti-inflammatory
cytokines from day 4 after MI [108]. Although macrophages had previously not been proven to
express the APJ receptor [109], studies from recent years have yielded findings to the contrary [69,
110]. In murine models, apelin has been shown to inhibit monocyte recruitment in abdominal aorta
[69] and in kidney during diabetic nephropathy [111]. However, apelin has been reported to promote
monocyte adhesion to HUVECs via VCAM-1 [112]. Apelin has been found also to induce lower
production of pro-inflammatory cytokines in macrophages (IL-6, IL-8 MIP-1α, M-CSF) and to reduce
MCP-1 and TNF-α production [69]. Apelin-mediated inhibition of MCP-1 and IL-8 production has been
reported also in peritoneal macrophages [113]. In view of the fact that there exist two
 ACCEPTED MANUSCRIPT

monocyte/macrophage subpopulations, we might assume that the CD16high subset, active later after
MI, expresses a higher level of APJ receptor after hypoxia stimulation and hence responds to apelin
more effectively. This in effect leads to a reduction of inflammatory cytokines and promotes
pro-angiogenic behaviour of the monocytes/macrophages in the later phase after MI. (Figure 3)

8. Apelin in acute myocardial infarction and its therapeutic potential

T
8.1 From animal models to human studies

P
Plasma level of apelin after acute myocardial infarction (AMI) is lower than in healthy people or in

RI
patients with coronary artery disease [114]. Nevertheless, apelin concentration rises over time after
AMI [115]. Additional apelin induction seems to improve blood vessel formation. Animal experiments

SC
help us to better understand the mechanisms and impact of apelin treatment, and at least partially
predict its effect in human. Majority of in vivo experiments have been performed in rodents. Apelin is
mostly administered intravenously (i.v.) or by intraperitoneal route (i.p.) [116, 117] in its
pyroglutamated form [Pyr1]-apelin-13, and its dosage varied from nanomolar to micromolar

NU
concentrations per kilogram. Apelin treatment usually starts at day of surgery or several days before
and continues up to 14 days after surgery [117]. Despite variances in protocols of apelin
administration, most of the studies coincide on apelin ability to reduce fibrosis and increase cardiac
MA
contractility and capillary density. In recent study post-infarct treatment with [Pyr1]-apelin-13 for 5
days increased heart rate to the level of control group in comparison with MI rats without apelin
[116]. However, systolic blood pressure, diastolic blood pressure and mean arterial pressure (MAP)
were not affected. Importantly, apelin reduced infarct size and increased nitric oxide (NO) level [116].
ED

The same group also reported reduction of fibrosis and increased capillary density in myocardium
after [Pyr1]-apelin-13 treatment in rats [118]. In mouse experiment, apelin-13 treatment post MI
decreased fibrosis, the area of MI (assessed by the ratio of the infarcted area to left ventricle area)
PT

and increased capillary density and cardiac contractility [117]. There are rare reports on apelin
administration in large animal models. In sheep, three different doses of apelin in i.v. bolus were
compared, but only the highest dose of 1mg (equivalent of 20µg/kg) had significant impact on MAP
CE

and heart rate. However, these changes were short-term, with return to control level within 15-20
min. More importantly, atrioventricular block lasting 15 min was observed in several sheep [119].
Recently, escalating administration of apelin to dogs with heart failure for four hours has been
AC

studied. Dogs received apelin for two hours in low dose (500ng/kg/min) followed by two hours in
high dose (50µg/kg/min). Apelin treatment improved left ventricle (LV) systolic function with no
effect on heart rate [120].

Apelin carried by nanoparticles could represent novel therapeutic approach in AMI. Serpooshan et al.
[121] have prepared liposomal particles with encapsulated [Pyr1]-apelin-13 and coated with
polyethylene glycol (PEG) polymer. Apelin loaded nanoparticles were administered to mice after
transverse aortic constriction with single injection a week for two weeks. At days 8 and 14 post-
surgery cardiac contractility was significantly improved and hypertrophic remodelling was inhibited
in comparison with control group treated only with saline. In contrast, administration of regular
[Pyr1]-apelin-13 by intraperitoneal injection had lower effect on hypertrophic response. Also in heart
sections, [Pyr1]-apelin-13 encapsulated in nanoparticles reduced fibrosis extent and left ventricle size
more efficiently than regular [Pyr1]-apelin-13 [121]. Another possible way of AMI treatment with
growth factors is gene transfer. Apelin gene transfer in the hind limb ischemia model has been shown
to reduce necrosis, and the combination of apelin and VEGF gene therapy was even more effective
[18]. While VEGF gene transfer alone increased the number of blood vessels, apelin was found to
induce fewer but larger vessels. In the combination therapy, apelin inhibited VEGF-mediated
leakiness of blood vessels [18]. Vascular leakage depends on VE-cadherin-mediated EC–EC junctions,
 ACCEPTED MANUSCRIPT

and it is known that VEGF stimulation leads to loss of VE-cadherin and destroys the junctions [122].
Apelin has been found to abolish the effect of VEGF on gap formation, but it does not affect cell
junctions directly [18]. The molecular mechanism of this process is not yet clear, however, and
possible cross-linking with the Ang-1 pathway has been suggested [18].

As animal models do not reflect sufficiently the situation in human, human studies are essential to

T
correctly evaluate the efficiency and safety of the drug. Apelin effect in human depends not only on
way of administration and dosage, but also differs in healthy people and patients with heart failure.

P
In recent human study the effect of apelin-36 and [Pyr1]-apelin-13 was explored in different

RI
protocols [123]. In volunteers, the effect of apelin-36 on coronary artery and myocardial contractility
was evaluated. Intracoronary boluses of apelin-36 and saline were administered via a coronary guide
catheter in two different doses (20 and 200nmol in 2ml). While there were no changes in heart rate

SC
and blood pressure in both doses, apelin-36 administration in higher dose (200nmol) increased
coronary blood flow [123]. In the second protocol, intrabrachial administration of apelin caused
vasodilatation both in volunteers and in patients with heart failure. Apelin systemic administration

NU
into large antecubital veins of healthy volunteers in high dose led to slightly increased heart rate,
increased cardiac output, and reduced peripheral blood resistance. In contrast, in patients with heart
failure increase in cardiac output and reduction in blood pressure and peripheral blood resistance
MA
could be seen. It was also found that apelin effect on blood pressure depends on age. In young
healthy subjects was blood pressure maintained, whereas older subjects and patients with heart
failure had slight reduction [123].
ED

8.2 Apelin analogues can overcome apelin short half-life

The use of native apelin peptide for in vivo treatment is hampered by its short half-life, which is not
longer than 5 min [124-126] and is particularly caused by rapid proteolysis by angiotensin-converting
PT

enzyme 2 (ACE2) [124]. To overcome this drawback, apelin analogues with modified chemical
structure are being designed. To date, several synthetic apelin analogues have been prepared. One of
the first attempts was cyclic analogue derived from apelin-12 [127]. Apelin-12 analogues were also
CE

synthesized by Pisarenko et al. and administered by i.v. bolus injection at the onset of reperfusion
after left anterior descending (LAD) coronary artery occlusion at previously optimized dose of 0,35
µmol/kg. Apelin analogue with replaced methionine by nor-leucine and included Nα-methylarginine
AC

moiety in N-terminus of the peptide proved to reduce infarct size [128]. Apelin analogue II, more
resistant to proteolytic cleavage, was synthesized by Wang and his colleagues and applied ex vivo
after the ischemic period to damaged heart of APLN-/y and APLN+/y mice. This resulted in recovery of
myocardial performance and increased Akt and Erk1/2 phosphorylation [20]. The apelin analogue
also stimulated endothelial sprouting with longer capillary tube formation and increased phospho-
Akt in endothelial progenitor cells [20]. On the other hand, apelin deficiency in APLN-/y mice was
connected with reduced nuclear translocation of HIF-1α, inability to upregulate VEGF and decreased
angiopoietin-2 level in infarcted myocardium [20]. Other approach is development of analogues that
stabilize APJ receptor in certain conformation after binding. These apelin analogues bind to the same
receptor, but activate different signalling pathways [129]. MM07 mimicking conformation of apelin-
13, for example, was designed to preferentially stimulate the G-protein pathway over β-arrestin and
to avoid β-arrestin-dependent cardiac hypertrophy [125]. This analogue was reported to increase
cardiac output dose-dependently, but without effect on blood pressure or heart rate [125].

9. Conclusions

Despite significant progress in therapy, AMI remains the leading cause of mortality in the developed
world. The re-vascularization potential of percutaneous coronary intervention, which is today an
 ACCEPTED MANUSCRIPT

established therapy for AMI, has been shown to be improved by subsequent stem cell
transplantation. Nevertheless, the outcomes of clinical studies on stem cell transplantation vary and
point to little long-term benefit [130]. New approaches are therefore being applied in experimental
models, including cell-sheet transplantation [131, 132], gene modification of stem cells [11, 133], and
co-delivery of stem cells with pro-angiogenic growth factors [13]. Recently, apelin, a critical enhancer
of angiogenesis, was reported to be reduced in patients after MI [20] while lower apelin level had an

T
inhibitory effect on vascular sprouting and myocardial angiogenesis [20]. Hence, a supportive therapy

P
with apelin and its analogues might bring a beneficial effect to AMI patients. The effect of apelin in
animal models has been investigated with promising results [18, 116]. Apelin also has been used for

RI
pre-treatment of murine bone marrow cells which later were delivered intramyocardially, thereby
resulting in increased angiogenesis and attenuated cardiac fibrosis formation [134]. As can be seen in

SC
animal and human studies, three main parameters are to be considered in apelin treatment, apelin
form, length of treatment and way of administration. All three principle apelin forms, apelin-13,
[Pyr1]-apelin-13 and apelin-36, have comparable efficacy in cardiovascular tissue [29]. Though, there

NU
are slight differences between them. Apelin-36, for example, acts slower [135] and apelin-13 post-
translational modification of the N-terminal glutamate to [Pyr1]-apelin-13 protects this isoform from
exopeptidase degradation [136]. Investigation of apelin treatment in long-term time horizon will be
also needed as initial benefits from short-time apelin administration may not reflect situation in
MA
heart weeks or months later. It is known that for example in VEGF treatment, stability of newly
formed vessels is dependent on VEGF continued stimulation for up to 4 weeks, otherwise the vessels
regress [137]. To assure long-time apelin effect, two solutions how to overcome its short half-life are
being proposed, synthetic analogues and apelin encapsulated in nanoparticles. The main difference is
ED

that apelin in its analogue form will be accessible immediately after administration, whereas
encapsulation in nanoparticles enables slow release for longer period, in case of poly(lactic-co-
glycolic acid) (PLGA) nanoparticles weeks or even months [138]. Release from PLGA nanoparticles is
PT

much slower than from liposomal nanoparticles that were reported to have peak of apelin release
between 15 and 20h [121]. Moreover, speed of PLGA nanoparticles degradation can be regulated by
their size and polymer composition [138, 139]. As patients have decreased apelin production up to
CE

24 weeks after AMI [140], combined administration of apelin analogues or apelin in liposomal
nanoparticles providing quick source in first days after MI, with use of PLGA nanoparticles sustaining
apelin release for longer period could be beneficial. Safety of long-term apelin administration,
AC

however, needs to be verified, too. Although the finding that administration of apelin does not
induce left ventricular hypertrophy is encouraging [141], several cases of atrioventricular block have
been reported in sheep [119] and pro-atherosclerotic effect of apelin in carotid ligation model urges
for further safety studies [102]. Besides in vivo studies of apelin treatment, its signalling pathway will
be no less important field in apelin research. Apelin affects many signalling molecules in endothelial
cells, nevertheless several studies indicate that apelin can both inhibit and stimulate a single
transcription factor. This may result not only from distinct signalling pathways downstream from
apelin/APJ signalling which affect the factor but also from reactions with other molecules inside the
endothelial cell. In conclusion, although apelin is indisputably an important enhancer of
angiogenesis, the apelin pathway must be thoroughly investigated if it is to fulfil its pro-angiogenic
potential in AMI therapy.

Acknowledgements

This work was financially supported by the European Regional Development Fund (project FNUSA-
ICRC, CZ.1.05/1.1.00/02.0123).
 ACCEPTED MANUSCRIPT

References

1. Leung, D.W., Cachianes, G., Kuang, W.J., Goeddel, D.V., Ferrara, N., 1989. Vascular endothelial
growth factor is a secreted angiogenic molecule. Science 246 (4935), 1306-1309. DOI:
10.1126/science.2479986.
2. Davis, S., Aldrich, T.H., Jones, P.F., Acheson, A., Compton, D.L., Jain, V., et al., 1996. Isolation of
Angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87 (7),

T
1161–1169. DOI: 10.1016/S0092-8674(00)81812-7.

P
3. Salcedo, R., Wasserman, K., Young, H.A., Grimm, M.C., Howard, O.M., Anver, M.R., et al., 1999.
Vascular endothelial growth factor and basic fibroblast growth factor induce expression of

RI
CXCR4 on human endothelial cells: in vivo neovascularization induced by stromal-derived factor-
1alpha. Am. J. Pathol. 154 (4), 1125–1135. DOI: 10.1016/S0002-9440(10)65365-5.

SC
4. Das, H., George, J.C., Joseph, M., Das, M., Abdulhameed, N., Blitz, A., et al., 2009. Stem cell
therapy with overexpressed VEGF and PDGF genes improves cardiac function in a rat infarct
model. PLoS One 4 (10), e7325. DOI: 10.1371/journal.pone.0007325.
5. Kim, S.W., Lee, D.W., Yu, L.H., Zhang, H.Z., Kim, C.E., Kim, J.M., et al., 2012. Mesenchymal stem

NU
cells overexpressing GCP-2 improve heart function through enhanced angiogenic properties in a
myocardial infarction model. Cardiovasc. Res. 95 (4), 495–506. DOI: 10.1093/cvr/cvs224.
6. Mathison, M., Gersch, R.P., Nasser, A., Lilo, S., Korman, M., Fourman, M., et al., 2012. In vivo
MA
cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the
infarcted myocardium with vascular endothelial growth factor. J. Am. Heart. Assoc. 1 (6),
e005652. DOI: 10.1161/JAHA.112.005652.
7. Ribatti, D., Baiguera, S., 2013. Phase II angiogenesis stimulators. Expert Opin. Investig. Drugs 22
(9), 1157–1166. DOI: 10.1517/13543784.2013.813933.
ED

8. Shudo, Y., Cohen, J.E., MacArthur, J.W., Atluri, P., Hsiao, P.F., Yang, E.C., et al., 2013. Spatially
oriented, temporally sequential smooth muscle cell-endothelial progenitor cell bi-level cell sheet
neovascularizes ischemic myocardium. Circulation 128 (11 Suppl 1), S59–68. DOI:
PT

10.1161/CIRCULATIONAHA.112.000293.
9. Carvalho, J.L., Braga, V.B., Melo, M.B., Campos, A.C., Oliveira, M.S., Gomes, D.A., et al., 2013.
Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction. J. Cell.
CE

Mol. Med. 17 (5), 617–625. DOI: 10.1111/jcmm.12036.

10. Lan, F., Liu, J., Narsinh, K.H., Hu, S., Han, L., Lee, A.S., et al., 2012. Safe genetic modification of
cardiac stem cells using a site-specific integration technique. Circulation 126 (11 Suppl 1), S20–
AC

28. DOI: 10.1161/CIRCULATIONAHA.111.084913.

11. Moon, H.H., Joo, M.K., Mok, H., Lee, M., Hwang, K.C., Kim, S.W., et al., 2014. MSC-based VEGF
gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated
polyethyleneimine. Biomaterials 35 (5), 1744–1754. DOI: 10.1016/j.biomaterials.2013.11.019.
12. Formiga, F.R., Pelacho, B., Garbayo, E., Imbuluzqueta, I., Díaz-Herráez, P., Abizanda, G., et al.,
2014. Controlled delivery of fibroblast growth factor-1 and neuregulin-1 from biodegradable
microparticles promotes cardiac repair in a rat myocardial infarction model through activation of
endogenous regeneration. J. Control. Release. 173, 132–139. DOI:
10.1016/j.jconrel.2013.10.034.
13. Wang, X., Li, Q., Hu, Q., Suntharalingam, P., From, A.H.L., Zhang, J., 2014. Intra-myocardial
injection of both growth factors and heart derived Sca-1+/CD31− cells attenuates post-MI LV
remodeling more than does cell transplantation alone: neither intervention enhances
functionally significant cardiomyocyte regeneration. PLoS One 9 (6), e95247. DOI:
10.1371/journal.pone.0095247.
14. Distler, J.H., Hirth, A., Kurowska-Stolarska, M., Gay, R.E., Gay, S., Distler, O., 2003. Angiogenic
and angiostatic factors in the molecular control of angiogenesis. Q. J. Nucl. Med. 47 (3), 149-161.
15. Lee, R.J., Springer, M.L., Blanco-Bose, W.E., Shaw, R., Ursell, P.C., Blau, H.M., 2000. VEGF gene
delivery to myocardium: deleterious effects of unregulated expression. Circulation 102 (8), 898–
901. DOI: 10.1161/01.CIR.102.8.898.
 ACCEPTED MANUSCRIPT

16. Ito, Y., Tsurushima, H., Sato, M., Ito, A., Oyane, A., Sogo, Y., et al., 2013. Angiogenesis therapy for
brain infarction using a slow-releasing drug delivery system for fibroblast growth factor 2.
Biochem. Biophys. Res. Commun. 432 (1), 182–187. DOI: 10.1016/j.bbrc.2013.01.013.
17. Yang, X., Zhu, W., Zhang, P., Chen, K., Zhao, L., Li, J., et al., 2014. Apelin-13 stimulates
angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in
myocardial microvascular endothelial cells. Mol. Med. Rep. 9 (5), 1590–1596. DOI:

T
10.3892/mmr.2014.1984.
18. Kidoya, H., Naito, H., Takakura, N., 2010. Apelin induces enlarged and nonleaky blood vessels for

P
functional recovery from ischemia. Blood 115 (15), 3166–3174. DOI: 10.1182/blood-2009-07-
232306.

RI
19. Kasai, A., Ishimaru, Y., Higashino, K., Kobayashi, K., Yamamuro, A., Yoshioka, Y., et al., 2013.
Inhibition of apelin expression switches endothelial cells from proliferative to mature state in

SC
pathological retinal angiogenesis. Angiogenesis 16 (3), 723–724. DOI: 10.1007/s10456-013-9349-
6.
20. Wang, W., McKinnie, S.M., Patel, V.B., Haddad, G., Wang, Z., Zhabyeyev, P., et al., 2013. Loss of

NU
Apelin exacerbates myocardial infarction adverse remodeling and ischemia-reperfusion injury:
therapeutic potential of synthetic Apelin analogues. J. Am. Heart. Assoc. 2 (4), e000249. DOI:
10.1161/JAHA.113.000249.
21. Zeng, H., He, X., Hou, X., Li, L., Chen, J.X., 2014. Apelin gene therapy increases myocardial
MA
vascular density and ameliorates diabetic cardiomyopathy via upregulation of sirtuin 3. Am. J.
Physiol. Heart. Circ. Physiol. 306 (4), H585-597. DOI: 10.1152/ajpheart.00821.2013.
22. Tatemoto, K., Hosoya, M., Habata, Y., Fujii, R., Kakegawa, T., Zou, M.X., et al., 1998. Isolation and
characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem.
ED

Biophys. Res. Commun. 251 (2), 471–476. DOI: 10.1006/bbrc.1998.9489.

23. Falcão-Pires, I., Leite-Moreira, A.F., 2005. Apelin: a novel neurohumoral modulator of the
cardiovascular system. Pathophysiologic importance and potential use as a therapeutic target.
Rev. Port. Cardiol. 24 (10), 1263–1276.
PT

24. Masri, B., Knibiehler, B., Audigier, Y., 2005. Apelin signalling: a promising pathway from cloning
to pharmacology. Cell Signal. 17 (4), 415–426. DOI: 10.1016/j.cellsig.2004.09.018.
25. Lee, D.K., Ferguson, S.S., George, S.R., O’Dowd, B.F., 2010. The fate of the internalized apelin
CE

receptor is determined by different isoforms of apelin mediating differential interaction with β-

arrestin. Biochem. Biophys. Res. Commun. 395 (2), 185–189. DOI: 10.1016/j.bbrc.2010.03.151.
26. Kawamata, Y., Habata, Y., Fukusumi, S., Hosoya, M., Fujii, R., Hinuma, S., et al., 2001. Molecular
AC

properties of apelin: tissue distribution and receptor binding. Biochim. Biophys. Acta 1538 (2–3),
162–171. DOI: 10.1016/S0167-4889(00)00143-9.
27. De Mota, N., Reaux-Le Goazigo, A., El Messari, S., Chartrel, N., Roesch, D., Dujardin, C., et al.,
2004. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through
inhibition of vasopressin neuron activity and vasopressin release. Proc. Natl. Acad. Sci. USA 101
(28), 10464–10469. DOI: 10.1073/pnas.0403518101.
28. Azizi, M., Iturrioz, X., Blanchard, A., Peyrard, S., De Mota, N., Chartrel, N., et al., 2008. Reciprocal
regulation of plasma apelin and vasopressin by osmotic stimuli. J. Am. Soc. Nephrol. 19 (5),
1015–1024. DOI: 10.1681/ASN.2007070816.
29. Maguire, J.J., Kleinz, M.J., Pitkin, S.L., Davenport, A.P., 2009. [Pyr1]apelin-13 identified as the
predominant apelin isoform in the human heart: vasoactive mechanisms and inotropic action in
disease. Hypertension 54 (3), 598–604. DOI: 10.1161/HYPERTENSIONAHA.
30. O’Carroll, A.M., Selby, T.L., Palkovits, M., Lolait, S.J., 2000. Distribution of mRNA encoding
B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in
brain and peripheral tissues. Biochim. Biophys. Acta 1492 (1), 72–80. DOI: 10.1016/S0167-
4781(00)00072-5.
31. Kleinz, M.J., Skepper, J.N., Davenport, A.P., 2005. Immunocytochemical localisation of the apelin
receptor, APJ, to human cardiomyocytes, vascular smooth muscle and endothelial cells. Regul.
Pept. 126 (3), 233–240. DOI: 10.1016/j.regpep.2004.10.019.
 ACCEPTED MANUSCRIPT

32. Kostopoulos, C.G., Spiroglou, S.G., Varakis, J.N., Apostolakis, E., Papadaki, H.H., 2014.
Adiponectin/T-cadherin and apelin/APJ expression in human arteries and periadventitial fat:
implication of local adipokine signaling in atherosclerosis? Cardiovasc. Pathol. 23 (3), 131–138.
DOI: 10.1016/j.carpath.2014.02.003.
33. Davis, G.E., Senger, D.R., 2005. Endothelial extracellular matrix: biosynthesis, remodeling, and
functions during vascular morphogenesis and neovessel stabilization. Circ. Res. 97 (11), 1093-

T
1107. DOI: 10.1161/01.RES.0000191547.64391.e3.
34. Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A., Abramsson, A., et al., 2003.

P
VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell. Biol. 161 (6),
1163–1177. DOI: 10.1083/jcb.200302047.

RI
35. del Toro, R., Prahst, C., Mathivet, T., Siegfried, G., Kaminker, J.S., Larrivee ,B., et al., 2010.
Identification and functional analysis of endothelial tip cell-enriched genes. Blood 116 (19),

SC
4025–4033. DOI: 10.1182/blood-2010-02-270819.
36. Kidoya, H., Ueno, M., Yamada, Y., Mochizuki, N., Nakata, M., Yano, T., et al., 2008. Spatial and
temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during

NU
angiogenesis. EMBO J. 27 (3) 522–534. DOI: 10.1038/sj.emboj.7601982.
37. Kidoya, H., Kunii, N., Naito, H., Muramatsu, F., Okamoto, Y., Nakayama, T., et al., 2012. The
apelin/APJ system induces maturation of the tumor vasculature and improves the efficiency of
immune therapy. Oncogene 31 (27) 3254–3264. DOI: 10.1038/onc.2011.489.
MA
38. Kawahara, H., Naito, H., Takara, K., Wakabayashi, T., Kidoya, H., Takakura, N., 2013. Tumor
endothelial cell-specific drug delivery system using apelin-conjugated liposomes. PLoS One 8 (6),
e65499. DOI: 10.1371/journal.pone.0065499.
39. Lindahl, P., Johansson, B.R., Levéen, P., Betsholtz, C., 1997. Pericyte loss and microaneurysm
ED

formation in PDGF-B-deficient mice. Science 277 (5323), 242–245. DOI:

10.1126/science.277.5323.242.
40. Sato, T.N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., et
al., 1995. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel
PT

formation. Nature 376 (6535), 70–74. DOI: 10.1038/376070a0.

41. Augustin, H.G., Koh, G.Y., Thurston, G., Alitalo, K., 2009. Control of vascular morphogenesis and
homeostasis through the angiopoietin-Tie system. Nat. Rev. Mol. Cell. Biol. 10 (3), 165–177. DOI:
CE

10.1038/nrm2639.
42. Kälin, R.E., Kretz, M.P., Meyer, A.M., Kispert, A., Heppner, F.L., Brändli, A.W., 2007. Paracrine
and autocrine mechanisms of apelin signaling govern embryonic and tumor angiogenesis. Dev.
AC

Biol. 305 (2), 599–614. DOI: 10.1016/j.ydbio.2007.03.004.

43. Rastaldo, R., Cappello, S., Folino, A., Berta, G.N., Sprio, A.E., Losano, G., et al., 2011. Apelin-13
limits infarct size and improves cardiac postischemic mechanical recovery only if given after
ischemia: tissue distribution and receptor binding. Am. J. Physiol. Heart. Circ. Physiol. 300 (6),
H2308–2315. DOI: 10.1152/ajpheart.01177.2010.
44. Kang, Y., Kim, J., Anderson, J.P., Wu, J., Gleim, S.R., Kundu, R.K., et al., 2013. Apelin-APJ signaling
is a critical regulator of endothelial MEF2 activation in cardiovascular development. Circ. Res.
113 (1), 22–31. DOI: 10.1161/CIRCRESAHA.
45. Kasai, A., Ishimaru, Y., Kinjo, T., Satooka, T., Matsumoto, N., Yoshioka, Y., et al., 2010. Apelin is a
crucial factor for hypoxia-induced retinal angiogenesis. Arterioscler. Thromb. Vasc. Biol. 30 (11),
2182–2187. DOI: 10.1161/ATVBAHA.110.209775.
46. Semenza, G.L., Nejfelt, M.K., Chi, S.M., Antonarakis, S.E., 1991. Hypoxia-inducible nuclear factors
bind to an enhancer element located 3′ to the human erythropoietin gene. Proc. Natl. Acad. Sci.
USA 88 (13), 5680–5684.
47. Cox, C.M., D’Agostino, S.L., Miller, M.K., Heimark, R.L., Krieg, P.A., 2006. Apelin, the ligand for
the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for
normal vascular development of the frog embryo. Dev. Biol. 296 (1), 177–189. DOI:
10.1016/j.ydbio.2006.04.452.
 ACCEPTED MANUSCRIPT

48. Ronkainen, V.P., Ronkainen, J.J., Hänninen, S.L., Leskinen, H., Ruas, J.L., Pereira, T., et al., 2007.
Hypoxia inducible factor regulates the cardiac expression and secretion of apelin. FASEB J. 21
(8), 1821–1830. DOI: 10.1096/fj.06-7294com.
49. Eyries, M., Siegfried, G., Ciumas, M., Montagne, K., Agrapart, M., Lebrin, F., et al., 2008. Hypoxia-
induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis.
Circ. Res. 103 (4), 432–440. DOI: 10.1161/CIRCRESAHA.108.179333.

T
50. Liu, D., Wang, J., Kinzel, B., Mueller, M., Mao, X., Valdez, R., et al., 2007. Dosage-dependent
requirement of BMP type II receptor for maintenance of vascular integrity. Blood 110 (5), 1502–

P
1510. DOI: 10.1182/blood-2006-11-058594.
51. David, L., Mallet, C., Keramidas, M., Lamandé, N., Gasc, J.M., Dupuis-Girod, S., et al., 2008. Bone

RI
morphogenetic protein-9 is a circulating vascular quiescence factor. Circ. Res. 102 (8), 914–922.
DOI: 10.1161/CIRCRESAHA.107.165530.

SC
52. Poirier, O., Ciumas, M., Eyries, M., Montagne, K., Nadaud, S., Soubrier, F., 2012. Inhibition of
apelin expression by BMP signaling in endothelial cells. Am. J. Physiol. Cell. Physiol. 303 (11),
C1139–1145. DOI:10.1152/ajpcell.00168.2012.

NU
53. Ricard, N., Ciais, D., Levet, S., Subileau, M., Mallet, C., Zimmers, T.A., et al., 2012. BMP9 and
BMP10 are critical for postnatal retinal vascular remodeling. Blood 119 (25), 6162–6171. DOI:
10.1182/blood-2012-01-407593.
54. Larrivée, B., Prahst, C., Gordon, E., del Toro, R., Mathivet, T., Duarte, A., et al., 2012. ALK1
MA
signaling inhibits angiogenesis by cooperating with the Notch pathway. Dev. Cell. 22 (3), 489–
500. DOI: 10.1016/j.devcel.2012.02.005.
55. Alastalo, T.P., Li, M., de Jesus Perez, V., Pham, D., Sawada, H., Wang, J.K., et al., 2011. Disruption
of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human
ED

pulmonary arterial EC survival. J. Clin. Invest. 121 (9), 3735–3746. DOI: 10.1172/JCI43382.
56. de Jesus Perez, V.A., Alastalo, T.P., Wu, J.C., Axelrod, J.D., Cooke, J.P., Amieva, M., et al., 2009.
Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-β-catenin and Wnt-
RhoA-Rac1 pathways. J. Cell. Biol. 184 (1), 83–99. DOI: 10.1083/jcb.200806049.
PT

57. Moser, M., Binder, O., Wu, Y., Aitsebaomo, J., Ren, R., Bode, C., et al., 2003. BMPER, a novel
endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling
and endothelial cell differentiation. Mol. Cell. Biol. 23 (16), 5664–5679. DOI:
CE

10.1128/MCB.23.16.5664-5679.2003.
58. Heinke, J., Juschkat, M., Charlet, A., Mnich, L., Helbing, T., Bode, C., et al., 2013. Antagonism and
synergy between extracellular BMP modulators Tsg and BMPER balance blood vessel formation.
AC

J. Cell. Sci. 126 (14), 3082–3094. DOI: 10.1242/jcs.122333.

59. Saharinen, P., Eklund, L., Miettinen, J., Wirkkala, R., Anisimov, A., Winderlich, M., et al., 2008.
Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix
contacts. Nat. Cell. Biol. 10 (5), 527–537. DOI: 10.1038/ncb1715.
60. Qin, D., Trenkwalder, T., Lee, S., Chillo, O., Deindl, E., Kupatt, C., et al., 2013. Early vessel
destabilization mediated by Angiopoietin-2 and subsequent vessel maturation via Angiopoietin-
1 induce functional neovasculature after ischemia. PLoS One 8 (4), e61831. DOI:
10.1371/journal.pone.0061831.
61. Scharpfenecker, M., Fiedler, U., Reiss, Y., Augustin, H.G., 2005. The Tie-2 ligand angiopoietin-2
destabilizes quiescent endothelium through an internal autocrine loop mechanism. J. Cell. Sci.
118 (4), 771–780. DOI: 10.1242/jcs.01653.
62. Takakura, N., Kidoya, H., 2009. Maturation of blood vessels by haematopoietic stem cells and
progenitor cells: involvement of apelin/APJ and angiopoietin/Tie2 interactions in vessel caliber
size regulation. Thromb. Haemost. 101 (6), 999–1005. DOI: 10.1160/TH08-06-0358.
63. Cushing, S.D., Berliner, J.A., Valente, J.A., Territo, M.C., Navab, M., Parhami, F., et al., 1990.
Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human
endothelial cells and smooth muscle cells. Proc. Natl. Acad. Sci. USA 87, 5134-5138.
64. Moldovan, N.I., Goldschmidt-Clermont, P.J., Parker-Thornburg, J., Shapiro, S.D., Kolattukudy,
P.E., 2000. Contribution of monocytes/macrophages to compensatory neovascularization: the
 ACCEPTED MANUSCRIPT

drilling of metalloelastase-positive tunnels in ischemic myocardium. Circ. Res. 87 (5), 378–384.

DOI: 10.1161/01.RES.87.5.378.
65. Hong, K.H., Ryu, J., Han, K.H., 2005. Monocyte chemoattractant protein-1-induced angiogenesis
is mediated by vascular endothelial growth factor-A. Blood 105 (4), 1405–1407. DOI:
10.1182/blood-2004-08-3178.
66. Caselli, E., Fiorentini, S., Amici, C., Di Luca, D., Caruso, A., Santoro, M.G., 2007. Human

T
herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant protein 1-
dependent capillary-like structure formation: role of the IKK/NF-κB pathway. Blood 109 (7),

P
2718–2726. DOI: 10.1182/blood-2006-03-012500.
67. Ma, J., Wang, Q., Fei, T., Han, J.D.J., Chen, Y.G., 2007. MCP-1 mediates TGF-β–induced

RI
angiogenesis by stimulating vascular smooth muscle cell migration. Blood 109 (3), 987–994. DOI:
10.1182/blood-2006-07-036400.

SC
68. Aplin, A.C., Gelati, M., Fogel, E., Carnevale, E., Nicosia, R.F., 2006. Angiopoietin-1 and vascular
endothelial growth factor induce expression of inflammatory cytokines before angiogenesis.
Physiol. Genomics 27 (1), 20–28. DOI: 10.1152/physiolgenomics.00048.2006.

NU
69. Leeper, N.J., Tedesco, M.M., Kojima, Y., Schultz, G.M., Kundu, R.K., Ashley, E.A., et al., 2009.
Apelin prevents aortic aneurysm formation by inhibiting macrophage inflammation. Am. J.
Physiol. Heart Circ. Physiol. 296 (5), H1329–1335. DOI: 10.1152/ajpheart.01341.2008.
70. Hara, C., Kasai, A., Gomi, F., Satooka, T., Sakimoto, S., Nakai, K., et al., 2013. Laser-induced
MA
choroidal neovascularization in mice attenuated by deficiency in the apelin-APJ system. Invest.
Ophthalmol. Vis. Sci. 54 (6), 4321–4329. DOI: 10.1167/iovs.13-11611.
71. Lu, Y., Zhu, X., Liang, G.X., Cui, R.R., Liu, Y., Wu, S.S., et al., 2012. Apelin-APJ induces ICAM-1,
VCAM-1 and MCP-1 expression via NF-κB/JNK signal pathway in human umbilical vein
ED

endothelial cells. Amino Acids 43 (5), 2125–3136. DOI: 10.1007/s00726-012-1298-7.

72. Lee, J.H., Kim, H., Woo, J.H., Joe, E.H., Jou, I., 2012. 5, 8, 11, 14-eicosatetraynoic acid suppresses
CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1
mRNA stability. J. Neuroinflammation 9, 34. DOI: 10.1186/1742-2094-9-34.
PT

73. Sanders, W.G., Morisseau, C., Hammock, B.D., Cheung, A.K., Terry, C.M., 2012. Soluble epoxide
hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory
effects of a soluble epoxide hydrolase inhibitor. Am. J. Physiol. Cell Physiol. 303 (3), C278–290.
CE

DOI: 10.1152/ajpcell.00386.2011.
74. Siragusa, M., Katare, R., Meloni, M., Damilano, F., Hirsch, E., Emanueli, C., et al., 2010.
Involvement of phosphoinositide 3-kinase γ in angiogenesis and healing of experimental
AC

myocardial infarction in mice. Circ. Res. 106 (4), 757–768. DOI:

10.1161/CIRCRESAHA.109.207449.
75. Tang, Y., Vater, C., Jacobi, A., Liebers, C., Zou, X., Stiehler, M., 2014. Salidroside exerts angiogenic
and cytoprotective effects on human bone marrow-derived endothelial progenitor cells via
Akt/mTOR/p70S6K and MAPK signalling pathways. Br. J. Pharmacol. 171 (9), 2440–2456. DOI:
10.1111/bph.12611.
76. Masri, B., Morin, N., Cornu, M., Knibiehler, B., Audigier, Y., 2004. Apelin (65-77) activates p70 S6
kinase and is mitogenic for umbilical endothelial cells. FASEB J. 18 (15), 1909–1911. DOI:
10.1096/fj.04-1930fje.
77. Berta, J., Hoda, M.A., Laszlo, V., Rozsas, A., Garay, T., Torok, S., et al., 2014. Apelin promotes
lymphangiogenesis and lymph node metastasis. Oncotarget 5 (12), 4426–4437.
78. Vinals, F., Chambard, J.C., Pouyssegur, J., 1999. p70 S6 kinase-mediated protein synthesis is a
critical step for vascular endothelial cell proliferation. J. Biol. Chem. 274 (38), 26776–26782. DOI:
10.1074/jbc.274.38.26776.
79. Cho, D.H., Choi, Y.J., Jo, S.A., Ryou, J., Kim, J.Y., Chung, J., et al., 2006. Troglitazone acutely
inhibits protein synthesis in endothelial cells via a novel mechanism involving protein
phosphatase 2A-dependent p70 S6 kinase inhibition. Am. J. Physiol. Cell Physiol. 291 (2), C317–
326. DOI: 10.1152/ajpcell.00491.2005.
 ACCEPTED MANUSCRIPT

80. Liu, L.Z., Zheng, J.Z., Wang, X.R., Jiang, B.H., 2008. Endothelial p70 S6 kinase 1 in regulating
tumor angiogenesis. Cancer Res. 68 (19), 8183–8188. DOI: 10.1158/0008-5472.CAN-08-0819.
81. Mitchelhill, K.I., Michell, B.J., House, C.M., Stapleton, D., Dyck, J., Gamble, J., et al., 1997.
Posttranslational modifications of the 5′-AMP-activated protein kinase β1 subunit. J. Biol. Chem.
272 (39), 24475–24479. DOI: 10.1074/jbc.272.39.24475
82. Nagata, D., Mogi, M., Walsh, K., 2003. AMP-activated protein kinase (AMPK) signaling in

T
endothelial cells is essential for angiogenesis in response to hypoxic stress. J. Biol. Chem. 278
(33), 31000–31006. DOI: 10.1074/jbc.M300643200.

P
83. Ouchi, N., Kobayashi, H., Kihara, S., Kumada, M., Sato, K., Inoue, T., et al., 2004. Adiponectin
stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt

RI
signalling in endothelial cells. J. Biol. Chem. 279 (2), 1304–1309. DOI: 10.1074/jbc.M310389200.
84. Attané, C., Daviaud, D., Dray, C., Dusaulcy, R., Masseboeuf, M., Prévot, D., et al., 2011. Apelin

SC
stimulates glucose uptake but not lipolysis in human adipose tissue ex vivo. J. Mol. Endocrinol.
46 (1), 21–28. DOI: 10.1677/JME-10-0105.
85. Xu, S., Han, P., Huang, M., Wu, J.C., Chang, C., Tsao, P.S., et al., 2012. In vivo, ex vivo, and in vitro

NU
studies on apelin's effect on myocardial glucose uptake. Peptides 37 (2), 320–326. DOI:
10.1016/j.peptides.2012.08.004.
86. Philippova, M., Joshi, M.B., Pfaff, D., Kyriakakis, E., Maslova, K., Erne, P., et al., 2012. T-cadherin
attenuates insulin-dependent signalling, eNOS activation, and angiogenesis in vascular
MA
endothelial cells. Cardiovasc. Res. 93 (3), 498–507. DOI: 10.1093/cvr/cvs004.
87. Krupinski, J., Abudawood, M., Matou-Nasri, S., Al-Baradie, R., Petcu, E.B., Justicia, C., et al., 2012.
Citicoline induces angiogenesis improving survival of vascular/human brain microvessel
endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1. Vasc. Cell
ED

4 (1), 20. DOI: 10.1186/2045-824X-4-20.

88. Liu, G., Han, J., Profirovic, J., Strekalova, E., Voyno-Yasenetskaya, T.A., 2009. Gα13 regulates
MEF2-dependent gene transcription in endothelial cells: role in angiogenesis. Angiogenesis 12
(1), 1–15. DOI: 10.1007/s10456-008-9123-3.
PT

89. Sturtzel, C., Testori, J., Schweighofer, B., Bilban, M., Hofer, E., 2014. The transcription factor
MEF2C negatively controls angiogenic sprouting of endothelial cells depending on oxygen. PLoS
One 9 (7), e101521. DOI: 10.1371/journal.pone.0101521.
CE

90. McKinsey, T.A., Zhang, C.L., Olson, E.N., 2000. Activation of the myocyte enhancer factor-2
transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-
3-3 to histone deacetylase 5. Proc. Natl. Acad. Sci. USA 97 (26), 14400–14405. DOI:
AC

10.1073/pnas.260501497.
91. Dekker, R.J., Boon, R.A., Rondaij, M.G., Kragt, A., Volger, O.L., Elderkamp, Y.W., et al., 2006. KLF2
provokes a gene expression pattern that establishes functional quiescent differentiation of the
endothelium. Blood 107 (11), 4354–4363. DOI: 10.1182/blood-2005-08-3465.
92. Song, Y., Li, X., Wang, D., Fu, C., Zhu, Z., Zou, M.H., et al., 2013. Transcription factor Krüppel-like
factor 2 plays a vital role in endothelial colony forming cells differentiation. Cardiovasc. Res. 99
(3), 514–524. DOI: 10.1093/cvr/cvt113.
93. Lin, Z., Natesan, V., Shi, H., Dong, F., Kawanami, D., Mahabeleshwar, G.H., et al., 2010. Krüppel-
like factor 2 regulates endothelial barrier function. Arterioscler. Thromb. Vasc. Biol. 30 (10),
1952–1959. DOI: 10.1161/ATVBAHA.110.211474.
94. Chandra, S.M., Razavi, H., Kim, J., Agrawal, R., Kundu, R.K., de Jesus Perez, V., et al., 2011.
Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension.
Arterioscler. Thromb. Vasc. Biol. 31 (4), 814–820. DOI: 10.1161/ATVBAHA.110.219980.
95. McLean, D.L., Kim, J., Kang, Y., Shi, H., Atkins, G.B., Jain, M.K., et al., 2012. Apelin/APJ signaling is
a critical regulator of statin effects in vascular endothelial cells--brief report. Arterioscler.
Thromb. Vasc. Biol. 32 (11), 2640–2643. DOI: 10.1161/ATVBAHA.
96. Kwon, I.S., Wang, W., Xu, S., Jin, Z.G., 2014. Histone deacetylase 5 interacts with Krüppel-like
factor 2 and inhibits its transcriptional activity in endothelium. Cardiovasc. Res. 104 (1), 127–
137. DOI: 10.1093/cvr/cvu183.
 ACCEPTED MANUSCRIPT

97. Wang, W., Ha, C.H., Jhun, B.S., Wong, C., Jain, M.K., Jin, Z.G., 2010. Fluid shear stress stimulates
phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and
eNOS. Blood 115 (14), 2971–2979. DOI: 10.1182/blood-2009-05-224824.
98. Hashimoto, T., Kihara, M., Ishida, J., Imai, N., Yoshida, S., Toya, Y., et al., 2006. Apelin stimulates
myosin light chain phosphorylation in vascular smooth muscle cells. Arterioscler. Thromb. Vasc.
Biol. 26 (6), 1267–1272. DOI: 10.1161/01.ATV.0000218841.39828.91.

T
99. Hofmann, A.D., Friedmacher, F., Takahashi, H., Hunziker, M., Gosemann, J.H., Puri, P., 2014.
Decreased apelin and apelin-receptor expression in the pulmonary vasculature of nitrofen-

P
induced congenital diaphragmatic hernia. Pediatr. Surg. Int. 30 (2), 197–203. DOI:
10.1007/s00383-013-3450-1.

RI
100. Li, L., Li, L., Xie, F., Zhang, Z., Guo, Y., Tang, G., et al., 2013. Jagged-1/Notch3 signaling
transduction pathway is involved in apelin-13-induced vascular smooth muscle cells

SC
proliferation. Acta Biochim. Biophys. Sin. (Shanghai) 45 (10), 875–881. DOI:
10.1093/abbs/gmt085.
101. Liu, Q.F., Yu, H.W., You, L., Liu, M.X., Li, K.Y., Tao, G.Z., 2013. Apelin-13-induced proliferation and

NU
migration induced of rat vascular smooth muscle cells is mediated by the upregulation of Egr-1.
Biochem. Biophys. Res. Commun. 439 (2), 235–240. DOI: 10.1016/j.bbrc.2013.08.051.
102. Kojima, Y., Kundu, R.K., Cox, C.M., Leeper, N.J., Anderson, J.A., Chun, H.J., et al., 2010.
Upregulation of the apelin-APJ pathway promotes neointima formation in the carotid ligation
MA
model in mouse. Cardiovasc. Res. 87 (1), 156–165. DOI: 10.1093/cvr/cvq052.
103. Heil, M., Ziegelhoeffer, T., Pipp, F., Kostin, S., Martin, S., Clauss, M., et al., 2002. Blood monocyte
concentration is critical for enhancement of collateral artery growth. Am. J. Physiol. Heart Circ.
Physiol. 283 (6), H2411–2419. DOI: 10.1152/ajpheart.01098.2001.
ED

104. Czepluch, F.S., Bernhardt, M., Kuschicke, H., Gogiraju, R., Schroeter, M.R., Riggert, J., et al., 2014.
In vitro and in vivo effects of human monocytes and their subsets on new vessel formation.
Microcirculation 21 (2), 148–158. DOI: 10.1111/micc.12100.
105. Okuno, Y., Nakamura-Ishizu, A., Kishi, K., Suda, T., Kubota, Y., 2011. Bone marrow-derived cells
PT

serve as proangiogenic macrophages but not endothelial cells in wound healing. Blood 117 (19),
5264–5272. DOI: 10.1182/blood-2011-01-330720.
106. Passlick, B., Flieger, D., Ziegler-Heitbrock, H.W., 1989. Identification and characterization of a
CE

novel monocyte subpopulation in human peripheral blood. Blood 74 (7), 2527–2534.

107. Draude, G., von Hundelshausen, P., Frankenberger, M., Ziegler-Heitbrock, H.W., Weber, C.,
1999. Distinct scavenger receptor expression and function in the human CD14(+)/CD16(+)
AC

monocyte subset. Am. J. Physiol. 276 (4), H1144–1149.

108. Nahrendorf, M., Pittet, M.J., Swirski, F.K., 2010. Monocytes: protagonists of infarct inflammation
and repair after myocardial infarction. Circulation 121 (22), 2437–2445. DOI:
10.1161/CIRCULATIONAHA.109.916346.
109. Choe, W., Albright, A., Sulcove, J., Jaffer, S., Hesselgesser, J., Lavi, E., et al., 2000. Functional
expression of the seven-transmembrane HIV-1 co-receptor APJ in neural cells. J. Neurovirol. 6
(Suppl. 1), S61–69.
110. Obara, S., Akifusa, S., Ariyoshi, W., Okinaga, T., Usui, M., Nakashima, K., et al., 2014.
Pyroglutamated apelin-13 inhibits lipopolysaccharide-induced production of pro-inflammatory
cytokines in murine macrophage J774.1 cells. Mod. Res. Inflamm. 3 (2), 59–66. DOI:
10.4236/mri. 2014.32007.
111. Day, R.T., Cavaglieri, R.C., Feliers, D., 2013. Apelin retards the progression of diabetic
nephropathy. Am. J. Physiol. Renal Physiol. 304 (6), F788–800. DOI:
10.1152/ajprenal.00306.2012.
112. Li, X., Zhang, X., Li, F., Chen, L., Li, L., Qin, X., et al., 2010. 14-3-3 mediates apelin-13-induced
enhancement of adhesion of monocytes to human umbilical vein endothelial cells. Acta Biochim.
Biophy.s Sin. (Shanghai) 42(6), 403-409.
113. Pan, C.S., Teng, X., Zhang, J., Cai, Y., Zhao, J., Wu, W., et al., 2010. Apelin antagonizes myocardial
impairment in sepsis. J. Card. Fail. 16 (7), 609–617. DOI: 10.1016/j.cardfail.2010.02.002.
 ACCEPTED MANUSCRIPT

114. Grzywocz, P., Mizia-Stec, K., Wybraniec, M., Chudek, J., 2014. Adipokines and endothelial
dysfunction in acute myocardial infarction and the risk of recurrent cardiovascular events. J.
Cardiovasc. Med. (Hagerstown) [Epub ahead of print]. DOI: 10.2459/JCM.0000000000000042.
115. Weir, R.A., Chong, K.S., Dalzell, J.R., Petrie, C.J., Murphy, C.A., Steedman, T., et al., 2009. Plasma
apelin concentration is depressed following acute myocardial infarction in man. Eur. J. Heart
Fail. 11 (6), 551–558. DOI: 10.1093/eurjhf/hfp043.

T
116. Azizi, Y., Faghihi, M., Imani, A., Roghani, M., Nazari, A., 2013. Post-infarct treatment with [Pyr1]-
apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide

P
enhancement in the rat model of myocardial infarction. Peptides 46, 76–82. DOI:
10.1016/j.peptides.2013.05.006.

RI
117. Li, L., Zeng., H., Chen, J.X., 2012. Apelin-13 increases myocardial progenitor cells and improves
repair postmyocardial infarction. Am. J. Physiol. Heart. Circ. Physiol. 303 (5), H605-618. DOI:

SC
10.1152/ajpheart.00366.2012.
118. Azizi, Y., Faghini, M., Imani, A., Roghani, M., Zekri, A., Mobasheri, M.B., et al., 2015. Post-infarct
treatment with [Pyr1]apelin-13 improves myocardial function by increasing neovascularization

NU
and overexpression of angiogenic growth factors in rats. Eur. J. Pharmacol. 761:101-108. DOI:
10.1016/j.ejphar.2015.04.034.
119. Charles, C.J., Rademaker, M.T., Richards, A.M., 2006. Apelin-13 induces a biphasic
haemodynamic response and hormonal activation in normal conscious sheep. J. Endocrinol. 189
MA
(3), 701-710.
120. Wang, M., Gupta, R.C., Rastogi, S., Kohli, S., Sabbah, M.S., Zhang, K., et al., 2013. Effects of acute
intravenous infusion of apelin on left ventricular function in dogs with advanced heart failure. J.
Card. Fail. 19 (7), 509-516. DOI: 10.1016/j.cardfail.2013.05.004.
ED

121. Serpooshan, V., Sivanesan, S., Huang, X., Mahmoudi, M., Malkovskiy, A.V., Zhao, M., et al., 2015.
[Pyr1]-Apelin-13 delivery via nano-liposomal encapsulation attenuates pressure overload-
induced cardiac dysfunction. Biomaterials 37, 289-298. DOI:
PT

10.1016/j.biomaterials.2014.08.045.
122. Gavard, J., Gutkind, J.S., 2006. VEGF controls endothelial-cell permeability by promoting the β-
arrestin-dependent endocytosis of VE-cadherin. Nat. Cell. Biol. 8 (11), 1223–1234. DOI:
CE

10.1038/ncb1486.
123. Japp, A.G., Cruden N.L., Barnes, G., van Gemeren, N., Mathews, J., Adamson, J., et al., 2010.
Acute cardiovascular effects of apelin in humans: potential role in patients with chronic heart
failure. Circulation 121 (16), 1818–1827. DOI: 10.1161/CIRCULATIONAHA.109.911339.
AC

124. Vickers, C., Hales, P., Kaushik, V., Dick, L., Gavin, J., Tang, J., et al., 2002. Hydrolysis of biological
peptides by human angiotensin‐converting enzyme‐related carboxypeptidase. J. Biol. Chem. 277
(17), 14838–14843.
125. Brame, A.L., Maguire, J.J., Yang, P., Dyson, A., Torella, R., Cheriyan, J., et al., 2015. Design,
characterization, and first-in-human study of the vascular actions of a novel biased apelin
receptor agonist. Hypertension 65 (4), 834-840. DOI: 10.1161/HYPERTENSIONAHA.114.05099.
126. Murza, A., Parent, A., Besserer-Offroy, E., Tremblay, H., Karadereye, F., Beaudet, N., et al., 2012.
Elucidation of the structure-activity relationships of apelin: influence of unnatural amino acids
on binding, signaling, and plasma stability. ChemMedChem 7 (2), 318–325. DOI:
10.1002/cmdc.201100492.
127. Hamada, J., Kimura, J., Ishida, J., Kohda, T., Morishita, S., Ichihara, S., et al., 2008. Evaluation of
novel cyclic analogues of apelin. Int. J. Mol. Med. 22 (4), 547 – 552.
128. Pisarenko O.I., Serebryakova, L.I., Studneva, I.M., Pelogeykina, Y.A., Tskitishvili O.V., Bespalova
Z.D., et al., 2013. Effects of structural analogues of apelin-12 in acute myocardial infarction in
rats. J. Pharmacol. Pharmacother. 4 (3), 198-203. DOI: 10.4103/0976-500X.114600.
 ACCEPTED MANUSCRIPT

129. Ceraudo, E., Galanth, C., Carpentier, E., Banegas-Font, I., Schonegge, A.M., Alvear-Perez, R., et
al., 2014. Biased signaling favoring gi over β-arrestin promoted by an apelin fragment lacking the
C-terminal phenylalanine. J. Biol. Chem. 289 (35), 24599-24610. DOI: 10.1074/jbc.M113.541698.
130. Pavo, N., Charwat, S., Nyolczas, N., Jakab, A., Murlasits, Z., Bergler-Klein, J., et al., 2014. Cell
therapy for human ischemic heart diseases: Critical review and summary of the clinical
experiences. J. Mol. Cell. Cardiol. 75, 12–24. DOI: 10.1016/j.yjmcc.2014.06.016.

T
131. Alshammary, S., Fukushima, S., Miyagawa, S., Matsuda, T., Nishi, H., Saito, A., et al., 2013.

P
Impact of cardiac stem cell sheet transplantation on myocardial infarction. Surg. Today 43 (9),
970–976. DOI: 10.1007/s00595-013-0528-2.

RI
132. Tano, N., Narita, T., Kaneko, M., Ikebe, C., Coppen, S.R., Campbell, N.G., et al., 2014. Epicardial
placement of mesenchymal stromal cell-sheets for the treatment of ischemic cardiomyopathy;

SC
in vivo proof-of-concept study. Mol. Ther. 22 (10), 1864–1871. DOI: 10.1038/mt.2014.110.
133. Ong, S.G., Lee, W.H., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., et al., 2014. Cross talk of
combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.

NU
Circulation 130 (11 Suppl. 1), S60–69. DOI: 10.1161/CIRCULATIONAHA.113.007917.
134. Li, L., Zeng, H., Hou, X., He, X., Chen, J.X., 2013. Myocardial injection of apelin-overexpressing
bone marrow cells improves cardiac repair via upregulation of Sirt3 after myocardial infarction.
PLoS One 8 (9), e71041. DOI: 10.1371/journal.pone.0071041.
MA
135. Japp, A.G., Cruden, N.L., Amer, D.A, Li, V.K., Goudie, E.B., Johnston, N.R., et al., 2008. Vascular
effects of apelin in vivo in man. J. Am. Coll. Cardiol. 52 (11), 908-913. DOI:
10.1016/j.jacc.2008.06.013.
136. Van Coillie, E., Proost, P., Van Aelst, I., Struyf, S., Polfliet, M., De Meester, I., et al., 1998.
ED

Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the
amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry
37 (36), 12672-12680.
PT

137. Dor, Y., Djonov, V., Abramovitch , R., Itin, A., Fishman G.I., Carmeliet, P., et al., 2002. Conditional
switching of VEGF provides new insights into adult neovascularization and pro-angiogenic
therapy. EMBO J 21 (8), 1939-1947.
CE

138. Makadia, H.K., Siegel, S.J., 2011. Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled
Drug Delivery Carrier. Polymers (Basel) 3 (3), 1377-1397. DOI: 10.3390/polym3031377.
139. Danhier, F., Ansorena, E., Silva, J.M., Coco, R., Le Breton, A., Préat, V., 2012. PLGA-based
AC

nanoparticles: an overview of biomedical applications. J. Control Release 161 (2), 505-522. DOI:
10.1016/j.jconrel.2012.01.043.
140. Weir, R.A., Chong, K.S., Dalzell, J.R., Petrie, C.J., Murphy C.A., Steedman, T., et al., 2009. Plasma
apelin concentration is depressed following acute myocardial infarction in man. Eur. J. Heart.
Fail. 11 (6), 551-558. DOI: 10.1093/eurjhf/hfp043.
141. Ashley, E.A., Powers, J., Chen, M., Kundu, R., Finsterbach, T., Caffarelli, A., et al., 2005. The
endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading
in vivo. Cardiovasc. Res. 65 (1), 73–82. DOI: 10.1016/j.cardiores.2004.08.018.

Legends to figures

Figure 1. (colour in print, 2-column)

Model of signalling pathways affecting apelin expression in endothelial cells (ECs). Hypoxia, Ang-1
and BMP-2 upregulate apelin in ECs. BMP-2 induces the formation of the PPARγ/β-catenin complex
which binds to PPARγ response element (PPRE) in the apelin gene (APLN). In contrast, BMP-9
 ACCEPTED MANUSCRIPT

represses apelin expression via SMAD proteins. Notch signalling co-operates with SMAD proteins,
inducing expression of UNC5B and VEGFR1 and inhibiting apelin.

Alk = activin receptor-like kinase; Ang-1 = angiopoietin-1; BMP = bone morphogenetic protein; BMPR
= bone morphogenetic protein receptor; HIF = hypoxia-inducible factor; HRE = hypoxia-responsive
element; Notch-ICD = Notch receptor intracellular domain; PPARγ = peroxisome proliferator-

T
activated receptors; PPRE = PPARγ response element; Tie-2 = tyrosine-protein kinase receptor;
UNC5B = unc-5 homolog B; VEGFR1 = vascular endothelial growth factor receptor 1.

P
Figure 2. (colour in print, 2-column)

RI
Assumed cross-talk of signalling pathways downstream of apelin/APJ signalling in endothelial cells

SC
(ECs). AKT/mTOR and ERK signalling pathways mediate the phosphorylation of p706S kinase, which
induces ECs proliferation. MCP-1, important for recruitment of monocytes into ischemic area, can be
repressed by apelin via Akt signalling. Akt blocks SMAD-mediated induction of MCP-1. In contrast,
apelin can stimulate MCP-1 expression via JNK and NF-κB. MEF-2, which affects EC proliferation and

NU
capillary tube formation, is expressed after apelin stimulation via AMPK and is repressed by HDACs.
MEF-2 further activates KLF-2, a factor vital for EC proliferation, angiogenesis and tight junction of
ECs. MEF-2 can be repressed by HDAC5. Interaction between HDACs and MEF-2/KLF-2 can be
MA
disrupted by HDACs phosphorylation. Apelin also stimulates Ang-1 expression, and Ang-1 can further
enhance apelin production.

AMPK = Adenosine monophosphate-activated kinase; Ang-1 = angiopoietin-1; APJ = angiotensin II

ED

protein J; eNOS = endothelial NO-synthase; ERK = extracellular-signal-regulated kinase; Gα/β/γ = G-

protein α/β/γ; HDAC-5 = histone deacetylases; JNK = c-Jun NH2-terminal kinase; KLF-2 = Krüppel-like
factor 2; MCP-1 = monocyte chemoattractant protein-1; MEF-2 = myocyte enhancer factor 2. mTOR =
PT

mammalian target of rapamycin; PI3K = PI3 kinase; p70S6K = p70S6 kinase.

Figure 3. (colour in print, 2-column)

CE

Apelin effect on monocytes/macrophages after myocardial infarction (MI). During the first four
days after MI (necrotic phase), the monocyte/macrophage subset of CD14highCD16low/negative
phenotype produces pro-inflammatory cytokines. Later, increasing apelin secretion by endothelial
cells (ECs) inhibits the production of pro-inflammatory cytokines and CD14highCD16low/negative
AC

monocytes/macrophages secrete anti-inflammatory cytokines.

APJ = angiotensin II protein J; IL = interleukin; MCP-1 = monocyte chemoattractant protein-1; M-CSF

= macrophage colony-stimulating factor 1; MIP-1α = macrophage Inflammatory Protein 1α; TNF-α =
tumour necrosis factor alpha; VLA-4 = very late antigen-4; VCAM-1 = vascular cell adhesion molecule
1.
 ACCEPTED MANUSCRIPT

TP
RI
SC
NU
MA
ED
PT

Figure 1
CE
AC
 ACCEPTED MANUSCRIPT

TP
RI
SC
NU
MA
ED
PT

Figure 2
CE
AC
 ACCEPTED MANUSCRIPT

TP
RI
SC
NU
MA
ED
PT

Figure 3
CE
AC
 ACCEPTED MANUSCRIPT

T
P
RI
SC
Graphical abstract

NU
MA
ED
PT
CE
AC