Current Hypertension Reports (2018) 20: 17

https://doi.org/10.1007/s11906-018-0798-6

PATHOGENESIS OF HYPERTENSION (W ELLIOTT AND R SANTOS, SECTION EDITORS)

Angiotensin-(1–7) and Alamandine on Experimental Models

of Hypertension and Atherosclerosis
Fernando Pedro de Souza-Neto 1 & Melissa Carvalho Santuchi 1 & Mario de Morais e Silva 1 &
Maria José Campagnole-Santos 1 & Rafaela Fernandes da Silva 1

Published online: 14 March 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Purpose of Review The purpose of this review was to summarize the current knowledge on the role of angiotensin-(1–7)
[Ang-(1–7)] and alamandine in experimental hypertension and atherosclerosis.
Recent Findings The renin-angiotensin system (RAS) is a very complex system, composed of a cascade of enzymes, peptides,
and receptors, known to be involved in the pathogenesis of hypertension and atherosclerosis. Ang-(1–7), identified and charac-
terized in 1987, and alamandine, discovered 16 years after, are the newest two main effector molecules from the RAS, protecting
the vascular system against hypertension and atherosclerosis.
Summary While the beneficial effects of Ang-(1–7) have been widely studied in several experimental models of hypertension,
much less studies were performed in experimental models of atherosclerosis. Alamandine has shown similar vascular effects to
Ang-(1–7), namely, endothelial-dependent vasorelaxation mediated by nitric oxide and hypotensive effects in experimental
hypertension. There are few studies on the effects of alamandine on atherosclerosis.

Keywords Renin-angiotensin system . Angiotensin-(1–7) . Mas receptor . Alamandine . MrgD receptor; hypertension .
Atherosclerosis

Introduction muscle cell (VSMC) phenotype, presence of vascular inflam-

There is a global epidemia of cardiovascular disease (CVD).
The number of cases of CVD increased to 17.3 million in
2013, according to the Global Burden of Disease Study
2013 [1]. Hypertension and atherosclerosis are major risk fac-
tors for CVD morbidity and mortality. Although considered as
independent risk factors, they are closely related and very
often occur simultaneously, affecting the cardiovascular sys-
tem and resulting in damage to various organs. Hypertension
and atherosclerosis share some common vascular events such
as endothelium dysfunction, alterations on vascular smooth
This article is part of the Topical Collection on Pathogenesis of
Hypertension
* Rafaela Fernandes da Silva
rfdasilva.ufmg@gmail.com
1 humans [7]. Ang II generated in the brain, kidney, and blood
Department of Physiology and Biophysics, Institute of Biological
Sciences, Federal University of Minas Gerais, Av. Antônio Carlos.
6627, Pampulha, Belo Horizonte, MG 31270-901, Brazil
mation and oxidative stress, and activation of the renin-
angiotensin system (RAS) [2, 3].
RAS is a very complex and dynamic system, composed of
a cascade of enzymes and peptides, important for many phys-
iological processes and involved in the pathogenesis of hyper-
tension and atherosclerosis [4–6]. Initially described as a hor-
monal system, it is now well known that many of the enzymes
and peptides can be produced locally in a variety of organs,
including blood vessels. Angiotensin II (Ang II) is the best-
studied peptide, considered as the major end effector molecule
of the classical RAS axis, composed of angiotensin-
converting enzyme (ACE), Ang II, and angiotensin receptor
type I (AT1R). The activation of ACE/Ang II/AT1R axis
participates on blood pressure increase, as many studies
have demonstrated that chronic pharmacological administra-
tion of ACE inhibitors and AT1R blockers lowers blood
pressure in several animal models of hypertension and in
vessels promotes hypertension involving a complex mecha-
nism, including transcriptional activation, reactive oxygen
17 Page 2 of 15
species (ROS) production, inflammation, and many other
cellular events. Ang II promotes atherosclerosis and partici-
pates in all phases of the disease, from the early lesion
formation, growth, progression, until plaque rupture [8, 9].
Most of the mechanisms described about the participation of
Ang II on atherosclerosis involve oxidative stress and in-
flammation [10].
In the last decades, new RAS components have been dis-
covered and characterized, changing the classical view of
RAS as a linear cascade. The existence of an alternative axis,
composed by angiotensin-converting enzyme 2 (ACE2),
Ang-(1–7), and Mas receptor (MasR), that counteract many
of the effects of ACE/Ang II/AT1R is now well accepted [4].
Indeed, the activation of ACE2/Ang-(1–7)/MasR by pharma-
cological treatments or by genetic modification has shown
antihypertensive and antiatherosclerotic effects in different
experimental disease models.
Finally, in 2013 the research group of Santos identified and
characterized the newest peptide of the RAS. The
heptapeptide Ala1-Ang-(1–7) was detected in plasma of rats
and humans, and in the aorta of mice and rats. Named as
alamandine, the chemical structure of this peptide is very sim-
ilar to Ang-(1–7), with only one amino acid residue differ-
ence, Ala1, in the N-terminal residue instead of Asp1 [11••].
In this pioneering discovery article, Lautner, Vilella, Fraga-
Silva et al. [11••] showed that alamandine, differently from
Ang-(1–7), acts preferentially by its binding to the MrgD re-
ceptor [11••]. In spite of that, alamandine presented similar
hypotensive effects to Ang-(1–7) in conscious spontaneously
hypertensive rats (SHR) and when microinjected into the cau-
dal ventrolateral medulla of anesthetized Fisher rats.
Thereafter, two additional studies confirmed the hypotensive
effects of alamandine. In regard to atherosclerosis, up to the
present, only two studies have been published, with distinct
outcomes that it will be further discussed in this review.
Thus, the aim of this review article is to summarize the
current knowledge about the role of the two major peptides
effectors from the novel protective RAS axes, Ang-(1–7) and
alamandine, in experimental hypertension and atherosclerosis.
Novel RAS Axes
RAS is an important endocrine, paracrine, and autocrine sys-
tem responsible for the control of blood pressure,
hydroelectrolyte balance, and maintenance of cardiovascular
and renal structures and functions [9]. The classical axis of this
system is initiated with angiotensinogen, a 411-amino acid
(50–60 kDa) 2-globulin, synthesized in the liver from a single
gene and released into the bloodstream or produced at tissue
level [12]. This macromolecule is the only precursor to all
components of RAS and is cleaved by renin-forming angio-
tensin I (Ang I) from its N-terminal portion [13].
Curr Hypertens Rep (2018) 20: 17
Renin (30–40 kDa), a monospecific aspartyl protease, is
synthesized, stored, and released in its active form or as
prorenin from juxtaglomerular cells in the kidneys [14].
Many studies have shown that renin can also be synthesized
in the collecting tubules and released into tubular fluid [15,
16]. In addition, there is evidence for intracellular production
of renin in the brain, heart, and adrenals [15, 17]. Interestingly,
prorenin has been identified as a hormonal component of
RAS, acting on receptors for prorenin in various tissues [18].
Ang I is a decapeptide with very weak biological activity
and serves as a precursor for the formation of Ang II, an
octapeptide hormone considered the main effector of RAS
[19]. ACE (150–180 kDa) cleaves the dipeptide histidyl-
leucine in the C-terminal portion of Ang II [20–22].
Chymase (35 kDa) is a peptidase that can also form Ang II
by hydrolyzing the Phe8-His9 bond of Ang I, in addition to
being capable of metabolizing Ang II itself and forming
Ang-(1–4) and Ang-(1–5) [23]. Ang II is the main active mol-
ecule from the classical RAS, interacting with AT1R and an-
giotensin receptor type II (AT2R) in blood vessels, heart, kid-
neys, and brain. The inappropriate overactivity of the Ang II/
AT1R interaction is involved in the genesis and progression of
hypertension and atherosclerosis [24–26].
The understanding of RAS has been expanded, as new
components have been discovered and characterized, such as
ACE2, Ang-(1–7) and MasR. In 1988, Santos and colleagues
showed for the first time the formation of Ang-(1–7) after the
incubation of dog brainstem homogenates with [125I]-Ang I,
in the absence or presence of ACE inhibitors [27••]. Thus,
these authors demonstrated that Ang-(1–7) was not only
formed by the metabolism of Ang II. In this same year,
Schiavone et al. [28] showed that Ang-(1–7) was able to in-
duce neurosecretory activity in the rat hypothalamo-
neurohypophysial system (HNS), promoting release of arginin
vasopressin (AVP) in vitro. Campagnole-Santos et al. [29]
also demonstrated that Ang-(1–7) promotes hypotensive re-
sponses when microinjected into the dorsal medulla of rats,
more specifically in the nucleus of the solitary tract. In addi-
tion to these evidences of the biological activity of Ang-(1–7)
in the nervous system, Chappel et al. identified the presence of
Ang-(1–7) in rat blood circulation and adrenals [30••].
Endogenous Ang-(1–7) can be formed directly from Ang I
by the hydrolysis of the Pro7-Phe8 peptide bond, in the C-
terminal portion by the enzymes prolyl oligopeptidase and
thimet oligopeptidase, besides neprilysin. In addition, the ac-
tion of ACE on Ang I produces Ang II, which subsequently
undergoes enzymatic action of ACE2 on the residue of Phe8 in
its C-terminal moiety to give rise to Ang-(1–7) [31]. ACE2
can also act on Ang I to form Ang-(1–9), which in turn is
converted to Ang-(1–7) [32].
In the year of 2003, Santos et al. identified Mas as the
functional receptor for Ang-(1–7). In this study, they demon-
strated that the antidiuretic effect elicited by Ang-(1–7) was
Curr Hypertens Rep (2018) 20: 17
abolished in MasR-knockout mice. It was also observed that
Ang-(1–7) promotes the release of arachidonic acid in cells
transfected with this receptor, but the use of D-Ala7-Ang-(1–7)
(A-779), a potent and selective MasR antagonist, blocks this
effect. Furthermore, dose-dependent relaxation of Ang-(1–7)
was abolished in MasR-deficient aortic rings, without interfering
with endothelium-dependent relaxation by acetylcholine [33••].
Different studies have shown that activation of the
ACE2/Ang-(1–7)/MasR axis counteracts with many of the car-
diovascular effects of ACE/Ang II/AT1R, including anti-inflam-
matory, antiproliferative, antifibrotic, antihypertrophic, and
vasodilatory mechanisms of action [34–36].
Other peptides are also formed within the RAS and produce
biological effects, such as angiotensin-(2–8) (Ang III) and an-
giotensin-(3–8) (Ang IV) [34]. In addition to these, other pep-
tides have been identified more recently, such as angiotensin A
(Ang A), a vasoconstrictor octapeptide similar to Ang II, that
exerts its effects through AT1R [37] and alamandine that can be
formed by the action of ACE2 on the octapeptide angiotensin A
or by thedecarboxylation of the N-terminal amino acid of
Ang-(1–7), Asp1, into Ala1 [11]. Alamandine can be formed
by the action of ACE2 on the octapeptide Ang A or by the
decarboxylation of the N-terminal amino acid of Ang-(1–7),
Asp1, into Ala1. Although the molecular difference between
Ang-(1–7) and alamandine is only one amino acid residue, it
was shown that the vasorelaxation and hypotensive effects of
alamandine in mouse isolated aortic rings and in anesthetized
rats, respectively, were not blocked by the MasR antagonist,
A-779. In addition, the vasorelaxation effect of alamandine
was not blocked in aortic rings from MasR-knockout mice
[11••]. In 2008, by using the Ang-(1–7) antagonist, D-Pro7-
Ang-(1–7), Gembardt et al. demonstrated that Ang-(1–7) also
binds poorly to Mas-related G protein-coupled type D. Due to
the high degree of similarity of Ang-(1–7) to alamandine, it was
hypothesized that the latter could also be a MrgD agonist [38•].
In the first experiment, the vasorelaxation response evoked by
alamandine in isolated aortic rings was totally blocked by D-
Pro7-Ang-(1–7). Further testing this hypothesis, the authors
showed that pre-incubation of arteries with β-alanine, an ago-
nist of the MrgD receptor, also blocked the effects of
alamandine. Finally, binding assays of alamandine in CHO
cells transfected with the MrgD receptor revealed a novel sig-
naling pathway for this newly discovered peptide [11••]. MrgD
belongs to a family of G protein-coupled receptors present in
nociceptors, skeletal muscle, heart, testis, and neurons in ro-
dents, primates, and humans [11••, ]. Since the discovery of
alamandine as the new ligand of the MrgD receptor, an increas-
ing number of studies have been published evidencing the pro-
tective effects of the activation of this pathway. Up to now, the
specific enzyme that acts to promote descarboxylation of the
angiotensin is still not known. Despite of that, the presence of
the peptides Ang A and alamandine in the circulation stimulates
the development of studies in order to establish where, when,
Page 3 of 15 17
and how they play a role in the pathophysiology of cardiovas-
cular diseases. Here, we will highlight the main results obtained
with the use of alamandine in experimental hypertension and
atherosclerosis.
Angiotensin-(1–7) and Hypertension
The axis formed by ACE2/Ang-(1–7)/MasR is an important
regulator of cardiovascular functions. Its effects on blood pres-
sure occur mainly through its direct actions in blood vessels,
kidneys, and brain (Fig. 1) [39]. Administration of Ang-(1–7)
in adult Akita mice (type I spontaneously diabetic animals)
over a period of 6 weeks was able to protect against elevated
systolic blood pressure (SBP), which was abolished by the
simultaneous administration of A-779 [40]. Kangussu et al.
demonstrated that intracerebroventricular administration of
Ang-(1–7) reduced systolic and diastolic blood pressure in
transgenic hypertensive rats (mRen2)27 [41]. In the same
study, it was demonstrated that Ang-(1–7) also improves car-
diac parameters related to the hypertension, such as reduction
of cardiomyocyte hypertrophy, total collagen deposition, and
type I collagen and fibronectin gene expressions, as well as
reduction of the ratio between cardiac sympathetic and para-
sympathetic tonus. Fall in blood pressure was also observed in
transgenic mice overexpressing human ACE2 selectively in the
brain [42–47]. Protective effects of Ang-(1–7) were also ob-
served in fructose-fed (FF) rats [48]. In animals subjected to
chronic intracerebroventricular infusion, this peptide presented
normalization of mean arterial pressure (MAP) and barorreflex
bradycardia sensitivity index. In addition, Ang-(1–7) reduced a
cardiac sympathetic tone to lower levels than controls and
fructose-fed rats [48•]. Similar effects were demonstrated in
the injection of Ang-(1–7) into the lateral ventricle of the
DOCA-salt rats’ brain [49]. In this study, central administration
of Ang-(1–7) reduced MAP levels, improved barorreflex bra-
dycardia sensitivity indexes, and reduced also the sympathetic
ratio/parasympathetic tones [49]. It was also demonstrated by
this same group that transgenic rats that present a lifetime in-
crease in circulating levels of Ang-(1–7) and TGR(A1-7)3292
are protected against exacerbated blood pressure elevation, car-
diac dysfunction, and fibrosis induced by DOCA-salt hyperten-
sion [50••].
The effects of Ang-(1–7) on the autonomic modulation
were also found by Bertagnolli et al.; they observed an in-
crease in heart rate (HR) variability as well as reduction of
low frequency and increase of high frequency in SHR submit-
ted to oral administration of Ang-(1–7) included in 2-
Hydroxypropyl-β-cyclodextrin (HPβCD) for 10 weeks [51].
These authors demonstrated that Ang-(1–7) reduced the diameter
of cardiomyocytes in hypertensive animals compared to their
controls. Corroborating with these results, Ang-(1–7) adminis-
tered subcutaneously over a 28-day period was able to prevent
17 Page 4 of 15 Curr Hypertens Rep (2018) 20: 17

Fig. 1 Role of angiotensin-(1–7)

and alamandine in different
organs in experimental
hypertension. The renin-
angiotensin system (RAS) is
critically involved in
pathophysiological changes in
different organs in hypertension.
Among them are the blood
vessels, heart, kidneys, and brain.
Angiotensin-(1–7) [Ang-(1–7)]
and alamandine through the
binding to Mas and Mas-related G
protein-coupled receptor member
D (MrgD) receptors, respectively,
promote protective actions for the
organism, decreasing progression
and attenuating damage caused
by elevated blood pressure,
usually characterized by fibrosis,
remodeling, inflammation, and
excessive vasoconstriction.
CSAR indicates cardiac
sympathetic afferent reflex;
RVLM, rostral ventrolateral
medulla; CVLM, caudal
ventrolateral medulla; RSNA,
renal sympathetic nerve activity

blood pressure rise and reduce sympathetic activity of the renal
nerve in Sprague-Dawley (SD) rats submitted to intermittent
chronic hypoxia [52]. In addition, these authors demonstrated
that Ang-(1–7) reduced renal oxidative stress in this model by
decreasing levels of malondialdehyde (MDA) and increasing
superoxide dismutase (SOD) and catalase (CAT) expressions,
significantly attenuating renal fibrosis in these animals.
When administered directly in the rostral ventrolateral me-
dulla (RVLM) of two-kidney, one clip (2K1C)-operated rats,
Ang-(1–7) promoted an increase in the renal sympathetic
nerve activity (RSNA) and MAP in 2K1C rats and increased
cardiac sympathetic afferent reflex and sympathetic output in
renovascular hypertension, effects that were mediated by
MasR [53]. The effect of Ang-(1–7) when microinjected in
RVLM was also observed in animals with stress-induced hy-
pertension. However, there were no differences in blood pres-
sure between these animals and their respective controls [54].
In contrast, microinjection of Ang-(1–7) into the basolateral
amygdala (BLA) in Wistar rats attenuated the increase of
blood pressure induced by acute emotional stress. This hypo-
tensive effect of Ang-(1–7) occurred in a concentration-
dependent manner and was blocked by A-779, indicating the
participation of MasR [55].
Interestingly, in hypertension induced by 2K1C surgery, an
exacerbation of the vascular effects of Ang-(1–7) occurred. In
this experimental model of hypertension, an increase in the
vasodilatory response promoted by Ang-(1–7) in the carotid
artery rings of hypertensive animals was observed, probably
related to the fact that in these animals Ang-(1–7) and MasR
expressions are increased in the carotid artery from 2K1C
[56].
It is noteworthy that the cardioprotective effects can occur
independently of the regulation of blood pressure [57••]. In the
DOCA-salt hypertension model, Grobe et al. [58] demonstrated
that Ang-(1–7) attenuated cardiac fibrosis without interfering
with blood pressure. In this animal model of hypertension, the
protective effects of Ang-(1–7) occur through the activation of
Ca2+ signaling pathways, reduction of hypertrophic markers, and
activation of the Akt signaling pathway, even under conditions of
moderately elevated blood pressure [57]. As previously men-
tioned, Ang-(1–7) is the major endogenous ligand of MasR.
Thus, blocking this receptor prevents Ang-(1–7) from promoting
its protective effects on hypertension. Intracerebroventricular ad-
ministration of A-779 in female SD rats with aldosterone/NaCl-
induced hypertension exacerbated the increase in MAP levels
and thus demonstrating the importance of endogenous central
Ang-(1–7) in protection against blood pressure elevation [59].
Subcutaneous administration of the MasR antagonist, A-779, in
Ang II-induced hypertension in SD rats increased recruitment of
macrophages and total collagen deposition in cardiac tissue. In
Curr Hypertens Rep (2018) 20: 17 Page 5 of 15 17

addition, A-779 increased the expression of pro-inflammatory Recently, a new non-peptidic MasR agonist, CGEN-856S,
genes (Gp91, MCP-1, ICAM-1, and caspase 3), and pro-fibrotic has been shown to have effects similar to those promoted by
markers (TGF-β, TIMP-2, and Bcl2). However, in this study Ang-(1–7). In addition to the endothelium-dependent
there were no differences in blood pressure between the group vasorelaxant effect on rat aortic rings, it has antiarrhythmogenic
subjected to Ang II and A-779 co-treatment when compared to effects and promotes reduction in blood pressure in SHR.
the group treated with Ang II alone [60]. A subcutaneous admin- CGEN-856S was also effective in attenuating cardiac remod-
istration of Ang-(1–7) for 4 weeks reduced SBP and diastolic eling in hypertensive rats or experimental model of cardiac
blood pressure (DBP) as well as the Heart Weight/Body Weight hypertrophy [70, 71]. Although it is well established that
(HW/BW) ratio, and reduced cardiomyocyte diameter and vasorelaxant effects of Ang-(1–7) are MasR dependent, recent-
perivascular fibrosis in the heart, an effect that was attributed to ly it was shown that acute bolus infusion of Ang-(1–7) reduces
the activation of the Ang-(1–7)/Sirt3 signaling pathway [61•]. MAP in wild-type mice but not in animals with gene deletion of
Also, in a mouse model of chronic infusion of Ang II over a 4- the MrgD receptor (MrgD Knockout). This result indicates that
week period, Lin et al. demonstrated that Ang-(1–7) also reduced Ang-(1–7) may also bind to the MrgD receptor [72].
cardiac fibrosis and cardiomyocyte diameter, accompanied by an
expressive decrease of the gene expression for atrial natriuretric
peptide (ANP) and skeletal α-actin (SAA), in addition to atten-
Alamandine and Hypertension
uated cardiac oxidative stress via increased SOD activity and
reduced MDA expression [62]. Furthermore, in vitro studies
In the pioneering study of Santos’s group, it was demon-
demonstrated that Ang-(1–7) promotes these cardioprotective
strated that oral administration of alamandine/HPβCD, at a
effects through the activation of the NO/cGMP pathway and
single dose, decreased the blood pressure of SHR. This
inhibition of the prohypertrophic calcineurin/NFAT signaling
was the first demonstration of a long-lasting hypotensive
cascade [63••].
effect of alamandine [11••]. In addition, these authors
Activation of the ACE2/Ang-(1–7)/MasR axis has also
showed that alamandine has pressor and depressor effects
been reported to be protective against the occurrence of stroke
when microinjected into the RVLM and caudal ventrolat-
[64, 65]. Regenhardt et al. demonstrated that central adminis-
eral medulla (CVLM) of Fisher rats, respectively, and these
tration of Ang-(1–7) increased the survival of stroke-prone
effects were blocked by the co-administration of D-Pro7-
spontaneously hypertensive rats (spSHRs) fed a high-salt diet,
Ang-(1–7). Alamandine also had a hypotensive effect
in addition to decreasing the number of subcortical hemor-
when administered to the CVLM of renal hypertensive
rhages [66•]. These authors also demonstrated that the central
rats, 2K1C. In this study, the hypotensive effects of
infusion of Ang-(1–7) improved the neurological status of the
alamandine were blocked by PD123319 and D-Pro7, but
animals and reduced the number of Iba-1-positive cells
not by losartan and A-779 [73].
(microglial marker), without effects on blood pressure and
In a more recent study, Hekmat et al. observed that
cardiac and renal damage parameters that naturally occur in
alamandine presents a biphasic cardiovascular response in
these animals.
2K1C hypertensive rats. [74]. Intravenous administration of
These data were reinforced by recent studies demonstrating
alamandine in animals with renovascular hypertension pro-
the beneficial effects of AVE0991 on blood pressure regula-
duced an AT1R mediated blood pressure pressor effect,
tion and other pathophysiological mechanisms related to car-
followed by a hypotensive response. The fall in blood pressure
diovascular disease. In 2R1C rats, a dose of 3.0 mg/kg of
was mediated, in part, by PD-123319 sensitive receptors.
AVE0991 over a 28-day period reduced MAP, an effect that
Further systematic studies are needed to confirm the binding
was not observed at a dose lower than 1.0 mg/kg. However,
affinity of alamandine to AT1R, AT2R, MasR, and MrgD and
both doses improved baroreflex sensitivity and reduced cardi-
to establish the relative role of the interaction of alamandine
ac fiber thickening [67]. AVE0991 also had mimetic effects on
with these receptors in vivo. Some effects of alamandine and
Ang-(1–7)-induced rat coronary vasodilation, an effect that
Ang-(1–7) on several animal models of hypertension are listed
occurred through the NO-guanylate cyclase pathway.
in Table 1.
Interestingly, this effect was lost in hearts of rats subjected to
coarctation of the abdominal aorta, and restored with the use
of the AT1R antagonist losartan [68]. The effects of AVE0991
on vascular tonus regulation were also observed when chron- Angiotensin-(1–7) and Atherosclerosis
ically administered in SD rats subjected to high salt diet. Oral
treatment with a dose of 10−7 mol/L during 3 days restored the Since the first animal model of atherosclerosis was conceived
acetylcholine-induced vasodilation in the mesenteric arteries and characterized by Anitschkow and Chalatow [75•], many
on high salt diet animals to levels similar to their respective advances in basic science have been made toward a better
controls, without effects on blood pressure [69]. understanding of its pathophysiology [76–78]. The
Table 1 ACE2/Ang-(1–7)/MasR, AVE0991, and alamandine/MrgD in experimental hypertension

Animal model Drug/target Outcomes References

Akita mice (SDA type I) 500 μg/kg/day of Ang-(1–7) administered subcutaneously over a 6-week ↓ SBP [40]
17 Page 6 of 15

period
(mRen2)27 ICV infusion of 200 ng/h of Ang-(1–7) ↓ SBP and DBP [41]
↓ Cardiomyocyte hypertrophy
↓ Collagen deposition
↓ Type I collagen
↓ Fibronectin
SHR Oral administration of 72 μg/kg−1/day−1 of Ang-(1–7)/HP-βCD Improve HR variability [51]
↓ Reduction of low frequency and increase of high frequency
↓ Diameter of cardiomyocytes
SD rats—intermittent chronic hypoxia 400 ng/kg−1/min−1 of Ang-(1–7) administered subcutaneously over a 28-day Prevent blood pressure rise [52]
period ↓ RSNA
↓ MDA
↑ SOD
↑ CAT
2K1C rats RVLM microinjection of Ang-(1–7) (0.3 nmol) ↑ RSNA [53]
↑ MAP
↑ CSAR
↑ Sympathetic output
SIH rats RVLM microinjection of Ang-(1–7) (100 pmol) ↑ MAP [54]
No differences between groups
2K1C rats Cumulative concentration-response curves ↑ Vasodilatory response promoted by Ang-(1–7) in the carotid artery [56]
Ang-(1–7)—0.1 nmol/L–1 μmol/L rings
Female SD rats—aldosterone/NaCl-induced A-779 ICV infusion ↑ Increase in MAP levels [59]
hypertension 60 ng kg−1 min−1
Ang II-induced hypertension in SD rats A-779 at 1 mg/day administered subcutaneously for 4 weeks ↑ Recruitment of macrophages [60]
↑ Total collagen deposition in cardiac tissue
↑ Gp91, MCP-1, ICAM-1, and caspase 3
↑ TGF-β, TIMP-2, and Bcl2
No differences on blood pressure
Ang II-induced hypertension in SD rats 400 ng/kg/min of Ang-(1–7) administered subcutaneously for 4 weeks ↓ SBP and DBP [61•]
↓ HW/BW ratio
↓ Reduced cardiomyocyte diameter
↓ Perivascular fibrosis in the heart
C57BL/6 mice and chronic infusion of Ang II Subcutaneous administration of 500 μg/kg/day of Ang-(1–7) for 4 weeks ↓ Cardiac fibrosis and cardiomyocyte diameter [62]
↓ ANP ↓ SAA
↑ SOD activity
↓ MDA expression
spSHRs ICV infusion of Ang-(1–7)—1.0 μg in 0.15 μL ↑ The survival [66•]
ACSF/h ↓ The number of subcortical hemorrhages
Improved the neurological status
↓ The number of Iba-1-positive cells
FF rats Chronic intracerebroventricular infusion of 200 ng/h of Ang-(1–7) for Normalization of MAP and barorreflex bradycardia sensitivity index [48•]
4 weeks ↓ Cardiac sympathetic tone and
↓ Sympathetic ratio/parasympathetic tones
DOCA-salt rats Chronic intracerebroventricular infusion of 200 ng/h of Ang-(1–7) for ↓ MAP [49]
17 days ↑ Baroreflex bradycardia sensitivity index
Curr Hypertens Rep (2018) 20: 17
Curr Hypertens Rep (2018) 20: 17 Page 7 of 15 17

2K1C two-kidney, one clip, (mRen2)27 transgenic hypertensive rats, CAT catalase, CSAR cardiac sympathetic afferent reflex, DBP diastolic blood pressure, HP-βCD HP-β cyclodextrin, HR heart rate, ICV
intracerebroventricular, MAP mean arterial pressure, MDA malondialdehyde, SBP systolic blood pressure, SD Sprague-Dawley, SDA spontaneously diabetic animals, SHR spontaneously hypertensive rats,
References

RSNA renal sympathetic nerve activity, SOD superoxide dismutase, RVLM rostral ventrolateral medulla, SIH stress-induced hypertension, spSHRs stroke-prone spontaneously hypertensive rats, FF
cardiovascular effects evoked by the RAS depend on the bal-
ance between the classical and protective arms of RAS in the

[11••]
[70]

[74]
[73]
[69]
[67]
vessel layers [79]. Thus, among other risk factors for athero-
sclerosis, RAS dysregulation and/or overactivity has been im-

Restored the acetylcholine-induced vasodilation in the mesenteric

plicated as one of the leading mechanisms in atherosclerosis,
participating in all phases of the disease, from plaque devel-
opment and progression until its rupture [80].

Biphasic reponses on blood pressure receptor mediated

Within the cardiovascular system, most of the RAS com-
ponents have been previously reported to be present in the
heart and blood vessels, including veins, aorta, and small re-
Endothelium-dependent vasorelaxant effect
↓ Sympathetic ratio/parasympathetic tones

sistance arteries [79]. Evidence of the existence of a vascular

tissue RAS includes the generation and expression of compo-
nents of both classical and protective arms in blood vessels,
↑ Baroreflex sensitivity and

such as renin, angiotensinogen, ACE, Ang I, Ang II, AT1R,

Antiarrhythmogenic effect
↓ Cardiac fiber thickening

AT2R, ACE2, Ang-(1–7), MasR, alamandine, and MrgD re-

ceptor [11••, 81–84]. In this topic, we focus on the importance
of the protective arms of RAS for atheroprotection and atten-
uation of atherosclerotic lesions in animal models and obser-
Outcomes

arteries

↓ MAP
↓ MAP

↓ MAP
↓ MAP

vations in humans, which may provide an insight into future

therapeutic approaches.
Zulli et al. [85] first identified both ACE2 and AT2R ex-
Administration of AVE0991 (10–7 M) during 3 days in the drinking water

pressions in macrophages and VSMC within the atheroscle-

rotic plaque in a New Zealand White (NZW) rabbit model of
Oral administration of 3.0 mg/kg of AVE0991 over a 28-day period

Intravenous administration of a single 75 μg/kg dose of alamandine

atherosclerosis. In fact, Tesanovic et al. [86••] noticed for the

first time, in atherosclerotic lesions of ApoE knockout (ApoE-
Oral administration of alamandine/HPβCD at a single dose

KO) mice fed a high-fat diet, that long-term Ang-(1–7) treat-

ment alone diminished the longitudinal development of the
CVLM microinjection of alamandine (4, 40 pmol)

lesions, which was totally and partially reversed by A-779

Acute infusion of graded doses of CGEN-856S

and PD-123319. As by that time, PD-123319 was considered

Cumulative concentration-response curves

as a selective AT2R antagonist, the authors discussed the po-

CGEN-856S—0.0001 to 1 μmol/L and

t e n t i a l c r o s s t a l k b e t w e e n M a s R a n d AT 2 R f o r
atheroprotection and vasoprotection [86••]. More recent stud-
ies have identified PD-123319 also as an MrgD receptor an-
tagonist; thus, we cannot exclude the possibility that
alamandine is formed from Ang-(1–7) in this model, and pro-
tective effects observed are partially mediated by alamandine/
fructose-fed rats, DOCA-salt deoxycorticosterone acetate salt rats

MrgD signaling activation [11••].

Drug/target

Dong et al. [87], in a similar rabbit model of Zulli et al. [85],

with a gene transfer-based strategy, injected a one-time infusion
of transfected plasmids for ACE2 (AdACE2), showing a reduc-
tion in the intimal vessel layer thickness and the intimal-to-
medial ratio. Additionally, they showed an alteration of angio-
tensin metabolism toward an increased ACE2 expression and
activity, suggesting that the manipulation of its expression may
delay and stabilize the atherosclerotic process [87]. These au-
thors also reported that the intimal macrophage content and
SD rats—high salt (HS) diet

MMP-3 and MMP-9 expression and activity were diminished

and, despite unaltered VSMC numbers, there was increased
Table 1 (continued)

collagen production, which altogether contributed to the stabi-

Animal model

lization of atherosclerotic lesions [87]. Furthermore, the

AdACE2 treatment approach conducted in ApoE-KO mice
2K1C rats
2K1C rats
2K1C rats

fed Western-type diet for a month lowered serum triglyceride,

SHR
SHR

lipid content, and monocyte/macrophage accumulation within

17 Page 8 of 15
the atherosclerotic plaque [88, 89]. In 2008, a study involving
elder human patients, most of them men at different stages of
atherosclerosis, was performed by an in situ binding tech-
nique, which indicated that ACE isoforms were expressed
at both transcript and protein levels in diseased and non-
diseased vessels [90], showing a similar pattern of expression
throughout the atherosclerosis process. ACE2 activity, how-
ever, was significantly diminished from early to stable lesion
and increased from stable to thrombus-containing lesions,
suggesting that the protective action of the contra-regulatory
axis of the RAS is somehow lost in that process. Using the
previously described rabbit in vivo model [87] in association
with in vitro HUVEC and VSMC cell cultures, Zhang et al.
[91] confirmed previous data obtained with ACE2 expression
by AdACE2 infusion in plaques [85], showing a reduction in
morphological parameters, and showed its occurrence in vitro
[87]. AdACE2 treatment also increased Ang-(1–7) expres-
sion both in vivo and in vitro while abrogating ACE and
AT1R expressions. Furthermore, AdACE2 decreased the ex-
pression of inflammatory mediators, such as MCP-1 in vitro,
and suppressed the growth of VSMC and lowered macro-
phage accumulation in neointima [91].
In a study from our group, using the mouse model of shear
stress-induced atherogenesis and plaque vulnerability in
ApoE-KO mice, we reported that a 3-week treatment with
Ang-(1–7)/HPβCD downregulates collagen, lipid, and im-
mune cell contents in atherosclerotic plaques; however, dis-
tinct shear stress-prone regions respond differently to the treat-
ment [92••]. In a closer examination of the inflammation pro-
cess within the atherosclerotic plaque, Silva et al. [93], using
standard diet-fed MasR/ApoE-double-knockout (DKO) mice,
observed that decreased fat mass and worsened dyslipidemia
resulted in an increased pro-atherogenic environment without
affecting the size of aortic root lesions, despite the reduced
leptin serum levels. The lipid metabolic alterations observed
in the DKO mice were partially inferred as one of the causes
for the absence of increased atherogenesis and the unaltered
intraplaque contents of leukocytes, VSMC and MMPs, except
for the macrophage accumulation, which was reduced [93].
However, using Mas/ApoE-DKO mice fed a high-fat Western
diet for 3 months, it was observed that the aortic lesion area in
DKO mice was bigger and presented more accumulation of
elicited mononuclear cells, which expressed higher levels of
pro-inflammatory interleukins than in ApoE-KO mice [94•].
The deficiency in the MasR in those animals also affected
macrophage gene expression, inducing a pro-inflammatory
phenotype in the aorta [94•]. Besides reporting the positive
effects of a 4-week Ang-(1–7) treatment on the size of athero-
sclerotic lesions, Yang et al. [95] observed an increase in col-
lagen and VSMC content combined with lower numbers of
inflammatory macrophages and pro-inflammatory cytokines
in ApoE-KO mice. In another study, it was reported that a
long-term Ang-(1–7) treatment was able to reduce MMP-8,
Curr Hypertens Rep (2018) 20: 17
a proatherogenic MMP, through suppression of p38 MAPK
activity but stated that lower collagen I production in VSMC
from ApoE-KO mice fed a high-fat diet helped in stabilizing
atherosclerotic plaques. [96]. Altogether, these results showed
the importance of the MasR in the negative regulation of the
mechanisms involved in the atherogenic process and the in-
fluence of the diet type in the facilitation of atherosclerosis.
Through the years, some Ang-(1–7) analogues have been
synthesized and used in experimental protocols, such as
AVE0991, CGEN-856S, and others [97]. In the literature, only
AVE0991 has been used to mimic Ang-(1–7) actions on ath-
erosclerotic models [98–100]. Using ApoE-KO mice fed with
chow diet in combination with AVE0991 treatment for
4 months, Toton-Zuranska et al. [98] reported that no changes
in cholesterol and triglycerides were observed in the plasma of
these animals, but AVE0991 was able to reduce the athero-
sclerotic lesion area in both en face and cross-section methods
of aortic roots. Regarding the evaluation of the plasma levels
of important pro-inflammatory indicators such as IL-6, MCP-
1, IL-12, and serum amyloid A (SaA), Jawien et al. [99] re-
ported that the treatment of ApoE-KO mice with AVE0991
was able to lower them, besides the area of lipid and athero-
sclerotic changes in the aorta and in the aortic root, and VSMC
and macrophage staining as compared to the control group
ApoE-KO mice after 4 months of treatment [101]. In the same
animal model, Olszanecki et al. [100] assessed Ang-(1–7) and
Ang II concentrations through Ang I metabolism by mass
spectrometry in an ex vivo experimental protocol, which dem-
onstrated that ApoE-KO mice naturally express higher levels
of Ang II, but not Ang-(1–7). It was shown that the increased
synthesis of Ang II in atherosclerotic thoracic aorta from
ApoE-KO mice was significantly reduced by treatment with
AVE0991, but no differences in Ang-(1–7) production were
observed with or without diet. Another insight into the role of
Ang-(1–7) in atherosclerotic plaques by orally administered
AVE0991 was provided by Skiba et al. [102•]. In female
ApoE-KO mice, AVE0991 treatment was able to reduce the
pro-inflammatory responses of macrophages and peripheral
blood monocytes, as well as macrophage and leukocyte infil-
tration within the plaques by inhibiting the production of pro-
inflammatory cytokines in the perivascular adipose tissue,
which also has a similar MasR expression as to the control
group. As previously reported, the atherosclerotic lesion area
in the aortic arch and thoracic aorta decreased [102•]. Figure 2
shows some of the effects mediated by protective arms of
RAS in experimental atherosclerosis.
Alamandine and Atherosclerosis
The recent discovery and characterization of alamandine and
its receptor MrgD added a new level of complexity to the
Curr Hypertens Rep (2018) 20: 17
Fig. 2 The effects of angiotensin-(1–7), AVE-0991, and alamandine on
the pathophysiology of atherosclerosis. Within the vascular wall, there is
a highly inflammatory and necrotic microenvironment, which contributes
to immune cell recruitment and migration, as well as increased lipid
accu mulation, low-density protein (LDL) oxidation, and
metalloproteinase (MMPs) activity. Altogether, these events induce
atherosclerosis, in which different cell types are involved, such as
macrophages, endothelial cells, and vascular smooth muscle cells
(VSMC) (left panel). Animal models have provided useful information
on how atherosclerosis arises and advances; and usage of renin-
angiotensin system (RAS) peptides or analogues, such as angiotensin-
(1–7) [Ang-(1–7)], AVE0991, and alamandine, has been helpful in
known RAS [11••]. Lautner, Vilella, Fraga-Silva et al. [11••]
showed that alamandine evokes similar central cardiovascular,
antihypertensive, and vasodilatory effects as to those by
Ang-(1–7). To date, there are only three reports on the effects
of alamandine in atherosclerotic plaques [103–105•].
Habiyakare et al. [103] first reported the presence of the
MrgD receptor in healthy and diseased aortae and its co-
localization with eNOS-positive cells and, possibly, in macro-
phages in the NZW rabbit model fed a 1-month high-fat diet.
They also observed that the vasodilatory effects of alamandine
were reduced in thoracic, carotid, renal, and iliac arteries [103].
Da Silva et al., using a cast device in ApoE-KO mice fed
Western-type diet, demonstrated that alamandine treatment
for 1 month reduced degranulation of neutrophils in vitro and
in vivo, besides decreasing the levels of circulating and
intraplaque MMP-9 [104••]. However, no changes in lipid,
macrophage, and neutrophil contents or the MrgD expression
at both mRNA and protein levels were observed, as well as
Page 9 of 15 17
stabilizing atherosclerotic plaques and in regulating some blood and
intraplaque atherosclerotic markers (right panel). The activation of the
protective arm of RAS by Ang-(1–7) and AVE-0991 through the Mas
receptor (MasR) reduces plaque morphological parameters and
inflammatory cytokine production, while increasing collagen and
angiotensin-converting enzyme 2 (ACE2) and Ang-(1–7) expressions
(in blue). Alamandine, a newly characterized RAS peptide, has shown
to have some similar effects as to Ang-(1–7) through Mas-related G
protein-coupled receptor member D (MrgD), but reports on the
induction of plasminogen activator inhibitor-1 (PAI-1) highlights the
demand that further investigation of this peptide role in atherosclerosis
is conducted (in black)
intraplaque collagen deposition, which suggests no ameliora-
tion of plaque structural stability [104••]. Uchiyama et al.
[105•], using Wistar rats fed a chow diet, showed that a 2-
day treatment with alamandine or Ang-(1–7) had opposing
effects on leptin expression and secretion dynamics.
Alamandine, through the MrgD, receptor also evoked con-
flicting effects over two pro-atherogenic proteins in vitro and
in vivo such as reduction of leptin expression and secretion in
adipocytes, but increased plasminogen activator inhibitor-1
(PAI-1) [105•]. It is possible that alamandine may act more
as a protector of the endothelial function than as a plaque
stabilizer.
The reports mentioned above have helped to identify the
dynamics of the newly characterized alamandine/MrgD path-
way in atherosclerosis, but further research is needed to better
address its role in the atherosclerotic process. Table 2 summa-
rizes the effects of Ang-(1–7), AVE0991, and alamandine on
experimental atherosclerosis.
Table 2 ACE2/Ang-(1–7)/MasR, AVE0991, and alamandine/MrgD in experimental atherosclerosis

Animal model Drug/target Outcomes References

NZW rabbit One-time infusion of AdACE2 suspension (2.5 × 109 pfu/mL) ↓ Intimal vessel layer thickness and intimal-to-medial ratio [87, 91]
↓ Growth of VSMC and neointimal macrophage accumulation
17 Page 10 of 15

↓MMP-3 and MMP-9 expression

↓ ACE and AT1R expressions
↑ ACE2 activity and collagen production
↑ ACE2 and Ang-(1–7) expressions

ApoE-KO mice One-time infusion of AdACE2 suspension (2.5 × 109 pfu/mL) ↓ Lipid and monocyte/macrophage accumulation [88, 89]
↓ Serum levels of MCP-1, VCAM-1 and IL-6
↑ Neovascularization

ApoE-KO mice Subcutaneously administered Ang-(1–7) (24 or 48 g/kg per hour, 1 month) ↓ Longitudinal development of the lesions [86••]

ApoE-KO mice Ang-(1–7) with or without other drugsVarious doses (100–400 ng/kg/min, ↑ Collagen and VSMC content [95]
1 month) ↓ Size of atherosclerotic lesion, numbers of inflammatory macrophages, and
pro-inflammatory cytokines

ApoE-KO mice Ang-(1–7) with or without other drugs (400 ng/kg/min, 1 month) ↓ MMP-8 activity and collagen I production in VSMC [96]

ApoE-KO mice + Ang-(1–7)/HPβCD by gavage (≈ 30 μg/kg/day of Ang-(1–7) for 5 days/week, ↓ Collagen, lipid, and immune cell contents [92••]
cast 3 weeks)
MasR/ApoE-DKO MasR deficiency ↓ Leptin serum levels and macrophage accumulation [93, 94•]
mice ↑ Size of aortic root lesions
↑ Aortic lesion area in DKO mice
↑ Accumulation of immune cells and levels of inflammatory interleukins
Absence of increased atherogenesis
Wistar rats Injection of Ang-(1–7)Various doses, 2 × 2 days, interval 24 h ↑ Leptin expression and secretion in adipocytes [105•]

ApoE-KO mice AVE0991 combined diet (0.58 μmol/kg b.w./day, 4 months) ↓ Pro-inflammatory cytokines in plasma [98–101]
↓ VSMC and macrophage staining
↓ Atherosclerotic lesion area
↓ Area of lipid changes in aorta
↓ Area of atherosclerotic changes
↓ Increased tissue synthesis of Ang II
No differences in tissue Ang-(1–7) levels
No change in cholesterol and triglycerides in blood

ApoE-KO mice AVE0991 combined diet (0.58 μmol/kg b.w./day, 1–3 months) ↓ Inflammatory response of macrophages and peripheral blood monocytes [102]
↓ Immune cells infiltration
↓ Production of pro-inflammatory cytokines in pVAT
↓ Atherosclerotic lesion area

ApoE-KO mice Subcutaneously administered alamandine (24 μg/kg/h, 1 month) ↓ Degranulation of neutrophils and levels of circulating and intraplaque MMP-9 [104••]
No changes in intraplaque collagen, lipid, macrophage, and neutrophil contents or
the MrgD expression

Wistar rats Injection of alamandineVarious doses (2 days, interval 24 h) ↓ Leptin expression and secretion in adipocytes [105•]
↑ PAI-1 expression in adipocytes
Curr Hypertens Rep (2018) 20: 17
Curr Hypertens Rep (2018) 20: 17
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflicts of interest relevant
to this manuscript. All the authors have declared that there is nothing to
disclosure.
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
the authors.
Abbreviations and Acronyms 2K1C, Two-kidney, one clip;
Ang-(1–7), Angiotensin-(1–7); A-779, D-Ala7-
Angiotensin-(1–7); ACE, Angiotensin-converting enzyme;
ACE2, Angiotensin-converting enzyme 2; AdACE2,
Transfected plasmids for ACE2; Ang A, Angiotensin A; Ang I,
Angiotensin I; Ang II, Angiotensin II; Ang III,
Angiotensin-(2–8); Ang IV, Angiotensin-(3–8); Ang-(1–4),
Angiotensin-(1–4); Ang-(1–5), Angiotensin-(1–5); Ang-(1–9),
Angiotensin-(1–9); ANP, Atrial natriuretric peptide; ApoE-
KO, ApoE knockout; AT1R, Angiotensin receptor type I;
AT2R, Angiotensin receptor type II; AVP, arginine vasopres-
sin; Bcl2, B-cell lymphoma 2; BLA, basolateral amygdala;
BW, body weight; CAT, Catalase; CHO cells, Chinese hamster
ovary cells; CVD, cardiovascular diseases; CVLM, caudal
ventrolateral medulla; DBP, Diastolic blood pressure; DKO,
Double-knockout; DOCA-salt, Deoxycorticosterone acetate
salt; D-Pro7Ang-(1-7), D-Pro7Angiotensin-(1-7); GMP,
Guanosine monophosphate; Gp91, subunit of phagocyte
NADPH oxidase; HF, High frequency; HNS, hypothalamo-
neurohypophysial system; HPβCD, 2-Hydroxypropyl-β-cy-
clodextrin; HR, Heart rate; HUVEC, Human umbilical vein
endothelial cells; HW, heart weight; ICAM-1, Intercellular
adhesion molecule-1; IL-6, Interleukin-6; IL-12, Interleukin-
12; MAP, Mean arterial pressure; MasR, Mas receptor; MCP-
1, Monocyte chemoattractant protein-1; MDA,
Malondialdehyde; MMPs, Matrix metalloproteinases; MMP-
3, Matrix metalloproteinases 3; MMP-8, Matrix metallopro-
teinases 8; MMP-9, Matrix metalloproteinases 9; (mRen2)27,
Hypertensive transgenic rats; MrgD, Mas-related G protein-
coupled type D; NFAT, Nuclear factor of activated T-cells; NO,
Nitric oxide; NZW, New Zealand white; p38 MAPK, p38
mitogen-activated protein kinases; PAI-1, Plasminogen acti-
vator inhibitor-1; RAS, Renin-angiotensin system; ROS,
Reactive oxygen species; RSNA, Renal sympathetic nerve
activity; RVLM, Rostral ventrolateral medulla; SAA,
Skeletal α-actin; SaA, Serum amyloid A; SBP, Systolic
blood pressure; SD, Sprague-Dawley; SHR, Spontaneous
hypertensive rats; SOD, Superoxide dismutase; TGF-1,
Transforming growth factor-1; TIMP-2, Tissue inhibitor of
metalloproteinases-2; VCAM-1, vascular cell adhesion
protein-1; VSMC, Vascular smooth muscle cells
Page 11 of 15 17
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