The tissue reninangiotensin system and intracellular signalling

Ingrid Fleming, Karin Kohlstedt and Rudi Busse

Purpose of review
The reninangiotensin system is not what it was, or for that
matter not necessarily where we thought it should be. For
example, there is a novel angiotensin I-metabolizing enzyme
that generates angiotensin 17 rather than angiotensin II.
Moreover, we are slowly realizing the importance of local
rather than circulating angiotensin II.
Recent findings
Rather than concentrating on the systemic renin
angiotensin system, recent work has concentrated on
elucidating the consequences of increasing angiotensin
II production within specific organs, such as the heart and
vasculature, as well as in the pancreas and in adipose
tissue. Inhibition of angiotensin II production either using
angiotensin-converting enzyme inhibitors or angiotensin
II receptor blockers not only reverses remodelling but also
increases tissue insulin sensitivity. Targeting the renin
angiotensin system clinically delays the onset of type
2 diabetes, but the mechanisms involved are not clearly
understood. Moreover, at least one other angiotensinconverting enzyme homologue (ACE2) plays a significant
role in the regulation of heart and kidney function, and as it
generates angiotensin 17 from angiotensin I, it is
proposed to counteract the detrimental effects associated
with the activation of the classic reninangiotensin system.
Summary
There is a need to re-evaluate the role(s) played by the
molecular components of the extended local renin
angiotensin system and their role in vascular disease and
type 2 diabetes.

Abbreviations
ACE
NO
PPARg
RAS

angiotensin-converting enzyme
nitric oxide
peroxisome proliferator-activated receptor gamma
reninangiotensin system

2006 Lippincott Williams & Wilkins

1062-4821

Introduction

Curr Opin Nephrol Hypertens 15:813. 2006 Lippincott Williams & Wilkins.

Circulating angiotensin II is the main effector of the

reninangiotensin system (RAS) and is involved in the
global regulation of sympathetic activation and blood
pressure as well as fluid and electrolyte balance. The
angiotensin-converting enzyme (ACE) is generally
assumed to play a role in the generation of the potent
pressor agent, angiotensin II, by catalysing the extracellular conversion of the decapeptide angiotensin I, which
has little if any effect on blood pressure. However, it is
not widely appreciated that although circulating angiotensin II levels decrease in response to acute ACE
inhibitor treatment [1], circulating angiotensin II levels
tend to increase in patients taking ACE inhibitors over
long periods [2,3]. Other peptidases that are able to
convert angiotensin I into angiotensin II and the serine
protease chymase are thought to be responsible for more
than 80% of the angiotensin II formation in the human
heart and more than 60% of that in the arteries [4,5]. Such
findings, particularly in the light of the experimental and
clinical evidence of the effectiveness of ACE inhibitor
therapy, led to the hypothesis that tissue ACE levels and
the concentrations of angiotensin II generated locally
within a specific tissue play a more important role in
the regulation of blood pressure and cardiovascular
homeostasis than previously thought.

Vascular Signalling Group, Institut fur Kardiovaskulare Physiologie, Johann

Wolfgang Goethe Universitat, Frankfurt am Main, Germany

The angiotensin-converting enzymes

Keywords
angiotensin-converting enzyme, bradykinin, type 2 diabetes

Correspondence to Ingrid Fleming, PhD, Vascular Signalling Group, Institut fur

Kardiovaskulare Physiologie, Johann Wolfgang Goethe Universitat, Theodor-SternKai 7, D-60590 Frankfurt am Main, Germany
Tel: +49 69 6301 6972; fax: +49 69 6301 7668;
e-mail: fleming@em.uni-frankfurt.de
Sponsorship: Work performed in the authors laboratory is supported by the
Deutsche Forschungsgemeinschaft (FL 364/1-2) and the European Vascular
Genomics Network, a network of excellence supported by the European
Communitys 6th Framework Programme (contract no. LSHM-CT-2003-503254).
Current Opinion in Nephrology and Hypertension 2006, 15:813

Two distinct forms of ACE (dipeptidyl-carboxypeptidase

I) are expressed in humans, a somatic form that is particularly abundant on the endothelial surface of lung
vessels (but which is also expressed in all other endothelial cell types as well as in some smooth muscle cells,
monocytes, T lymphocytes and adipocytes), and a smaller
germinal form that is found exclusively in the testis. Both
forms of ACE exist at the cell surface as ectoenzymes,
where they hydrolyse circulating peptides. A soluble
form of ACE, which is derived from the membranebound form through the action of the ACE secretase
[6], is also present in serum and other body fluids. All of
the ACE enzymes hydrolyse a spectrum of circulating

The tissue reninangiotensin system and intracellular signalling Fleming et al. 9

peptides and catalyse, for example, the inactivation of the

vasodilator peptides bradykinin and kallidin [7]. Several
of the effects of ACE inhibitors can be blocked by the B2
kinin receptor antagonist, icatibant, indicating that the
accumulation of bradykinin and not only the prevention
of angiotensin II formation may underlie the beneficial
effects of this class of compounds.
More recently, ACE was reported to possess a glycosylphosphatidylinositol hydrolase activity and to play a key
role in the cleavage of glycosylphosphatidylinositolanchored proteins such as TESP5 and PH-20 from the
sperm membrane of wild-type mice; the lack of this
activity accounts for the infertility of male ACE / mice
[8]. However, it is currently unclear whether or not
human somatic ACE also possesses this activity, and if
so whether or not ACE glycosylphosphatidylinositol
hydrolase activity remains intact after its C-terminal
cleavage from the cell membrane to generating the
circulating or soluble form of the enzyme.
Chymases are serine proteases belonging to the chymotrypsin family, and are found in mast cells in multiple
tissues and species. There are two main groups of chymases (a and b), but only a-chymase has been identified
in humans whereas multiple chymases of the b-type are
found in rodents. Human chymase is able to convert a
number of substrates, including angiotensin II from angiotensin I, IL-1b from its precursor and endothelin-1
from big endothelin, as well as to activate matrix metalloproteases [9]. When considering the generation of
angiotensin II, ACE is the most significant source in
rabbits, pigs and in the human lung, whereas in the
human heart and vasculature chymase generates more
angiotensin II than ACE. It is therefore not surprising
that chymase has been implicated in the pathogenesis of
cardiovascular diseases, particularly in cardiac hypertrophy, heart failure, atherosclerosis and restenosis [10].
ACE2 is a zinc metalloprotease that shares approximately
42% identity with the catalytic domain of somatic ACE,
which is expressed in vascular endothelial cells of the
heart, kidney and testis and can be secreted from cells
by the cleavage to the transmembrane domain [11,12].
ACE2 hydrolyses a spectrum of peptides, in particular
angiotensin I to angiotensin 19 and angiotensin II to
angiotensin 17. The enzyme can also cleave des-Arg9bradykinin, but does not hydrolyse bradykinin and is
insensitive to ACE inhibitors [1113]. Although the role
played by ACE2 in the metabolism of angiotensin I and II
in the human coronary circulation is controversial [14],
the fact that ACE2 metabolizes angiotensin II to give the
vasodilator angiotensin 17 has been interpreted to mean
that ACE2 provides a counterbalance, preventing the
deleterious consequences of the overactivity of the classic
RAS [15]. The relationship between ACE2 levels and

cardiac function remains to be clarified, because although

lentiviral delivery of ACE2 has recently been reported to
protect against angiotensin II-induced cardiac hypertrophy and fibrosis [16], the cardiac-specific overexpression
of ACE2 was associated with severe, progressive conduction and rhythm disturbances, ventricular tachycardia and
sudden death [17]. On the other hand, aging ACE2 /
mice develop a significant defect in both the speed and
the overall percentage of contraction [18].
Intriguingly, ACE2 was recently identified as a receptor
for the coronavirus that induces the severe acute respiratory syndrome [19,20]. ACE2 and the angiotensin II
receptor AT2 are reported to protect mice from the severe
acute lung injury induced by acid aspiration or sepsis,
whereas other RAS components, including ACE, angiotensin II and the AT1 receptor promote disease pathogenesis [21].

Which tissues possess a local renin

angiotensin system?
Experimental studies have suggested that ACE inhibitors
with high tissue affinity confer a greater degree of vascular RAS suppression than those with low tissue affinity,
despite similar suppression of the circulating RAS. Cells
and tissues not implicated in the classic RAS are now
known to possess all the molecular machinery necessary
to generate angiotensin II. Tissues classed as expressing
a functional local RAS express angiotensinogen, renin,
renin-binding protein, ACE, chymase, as well as the
angiotensin II receptors (AT1 and AT2) and secrete
angiotensin II.
The heart and the systemic vasculature

All the components of the RAS have been found in

cardiomyocytes as well as in endothelial cells and vascular
smooth muscle cells. These cell types are able to generate angiotensin II and pro-inflammatory/pro-atherosclerotic stimuli, such as high cholesterol and insulin,
are able to activate this local RAS to increase oxygenderived free radical production, and induce oxidative
stress.
There is increasing evidence that the local RAS may be
involved in the maintenance of cardiovascular structure
and repair. ACE levels are increased in many forms of
vascular and cardiac hypertrophy, and the administration
of ACE inhibitors has led to the regression of hypertrophy. This effect of ACE on vascular remodelling is
highlighted by the report that the in-vivo gene transfer of
ACE into the uninjured rat carotid artery results in the
development of vascular hypertrophy independent of
systemic factors and haemodynamic effects [22]. The
selective overexpression of ACE in the heart also results
in morphological changes in the atria, arrhythmia, and
sudden death [23].

10 Hormones, autacoids, neurotransmitters and growth factors

Macrophages

ACE is expressed in peripheral blood monocytes and in

tissue macrophages. In atherosclerotic human coronary
arteries, ACE immunoreactivity is associated with macrophages as well as with smooth muscle cells and
T lymphocytes [24,25]. Given that angiotensin II activates monocytes and stimulates the expression of
tissue factor as well as the release of pro-inflammatory
cytokines, the induction of ACE in monocytes probably
exacerbates inflammatory responses. ACE expression is
reported to be higher in ruptured plaques than in fibrosclerotic plaques, and in the former ACE is highly
expressed in macrophages accumulated around the attenuated fibrous cap. Such findings indicate that the presence of ACE within lesions, atheromatous plaques, and
ruptured plaques contributes greatly to the further progression of atherosclerosis, presumably via an increase
in vascular angiotensin II formation and the inactivation
of bradykinin [25].
Pancreas

Evidence exists for the presence of a discrete tissue RAS

in the human pancreas. However, this pancreatic system
is not solely localized within the vasculature, as AT1
receptors and (pro)renin were detected in the beta cells
of the islets of Langerhans as well as in endothelial cells
of the pancreatic vasculature, whereas (pro)renin messenger RNA was only detected in connective tissue
surrounding the blood vessels and in reticular fibres
within the islets [26,27]. The finding that a functional
RAS is present within the pancreas may go a long way to
accounting for the beneficial effects of ACE inhibitors
and angiotensin II receptor blocker therapy on the onset
of type 2 diabetes.
Adipose tissue

Human pre-adipocytes possess a complete functional

RAS, and undifferentiated pre-adipocytes as well as
immature adipocytes secrete angiotensin II [28]. The
expression of the RAS components seems to relate to
body weight because obese women are reported to have
higher circulating angiotensinogen, renin, aldosterone,
and ACE than lean women, and lower angiotensinogen
gene expression in adipose tissue. Weight reduction
(approximately 5%), on the other hand, reduced angiotensinogen, renin and aldosterone levels and decreased
ACE expression [29]. Although ACE / and AT1 /
mice have no obvious changes in fat deposition, an
ACE inhibitor and an AT1 receptor antagonist were
found to reduce adipocyte size and to increase insulin
sensitivity in SpragueDawley rats fed a fructose-rich
diet [30]. The increase in insulin sensitivity also fits with
a recent report [31], which found that in subjects with
essential hypertension and insulin resistance, RAS blockade with either an ACE inhibitor or an AT1 antagonist
increased the secretion of the insulin-sensitizing adipo-

kine, adiponectin. The effectiveness of the AT1 receptor

antagonist contrasts with a recent report [32], which
attributed a role for AT2 receptor-dependent angiotensin
II signalling in increasing adipose cell mass and glucose
intolerance. AT2 receptor-deficient mice were protected
against high fat diet-induced obesity and obesity-related
glucose intolerance.
Although there is evidence linking the local RAS in
adipocytes with adipocyte differentiation and insulin
resistance, it is currently not possible to speculate on
the molecular mechanisms that are involved, because
there seem to be marked differences in the responses
to angiotensin II in human and murine adipocytes, not to
mention the cell lines currently in routine use.

Intracellular signalling by the renin

angiotensin system: angiotensin versus
bradykinin
A great deal is known about the signalling events that
take place after the stimulation of AT1 and AT2 receptors. AT1 receptor activation is generally associated with
vasoconstriction, aldosterone and vasopressin release,
sodium and water retention and sympathetic facilitation,
as well as cell proliferation, left ventricular hypertrophy,
nephrosclerosis, vascular media hypertrophy, endothelial dysfunction and neointima formation. AT2 receptors
are more highly expressed during foetal development
than in adulthood, but are upregulated under pathological conditions. AT2 activation has been linked to nitric
oxide (NO) production and counteracts several of the
growth responses initiated by the AT1 and growth factor
receptors [33]. Angiotensin receptors can also be detected intracellularly, e.g. in the nucleus [34], as can angiotensinogen, renin and angiotensin II [35]. Intracellular
angiotensin II is suggested to regulate cell communication, gene expression, and cellular growth, via both
receptor-dependent and independent means, and probably originates from either the internalization of its
receptors or intracellular synthesis [36]. It is thus also
possible that angiotensin II generated locally within
the heart and vascular wall plays an important role in
the development of endothelial dysfunction and atherosclerosis.
ACE inhibitors have been clinically proved to lower
blood pressure, improve endothelial function and prevent
cardiovascular events, such as stroke and myocardial
infarction as well as heart failure [37], although angiotensin II levels are not actually decreased during longterm ACE therapy. Partly for this reason, and partly
because ACE is also kininase II, attention focused on
the effects of ACE inhibitors on bradykinin signalling.
Many of the effects of ACE inhibitors in animal models
and in humans, such as the increase in endothelial
NO synthase expression and improved vasodilator

The tissue reninangiotensin system and intracellular signalling Fleming et al. 11

responsiveness, can be inhibited by blocking the B2 kinin

receptor [38]. Activation of the B2 kinin receptor is linked
with antioxidant and anti-inflammatory actions, and protection against stroke, cardiovascular and renal disease.
Gene transfer of tissue kallikrein; the serine proteinase that produces bradykinin from its substrate kininogen, is reported to result in a prolonged reduction in
blood pressure, to attenuate hypertrophy and fibrosis
in the heart and kidney of several hypertensive animal
models, as well as to protect against cardiac remodelling
and restenosis. All these effects were sensitive to the B2
kinin receptor antagonist icatibant [39].
The angiotensin II and bradykinin signalling pathways,
however, appear to be inextricably linked, and the cardioprotective effects of an AT1 receptor antagonist are
reported to be diminished in B2 kinin receptor knockout
mice [40]. Crosstalk may also take place between B2 and
AT2 receptors, and AT2 receptor overexpression in vascular smooth muscle cells stimulates the production of
bradykinin, which in turn increases NO production and
elicits vasodilatation [41]. Moreover, the presence of AT2
receptors is required for the flow-dependent dilation
mediated by endogenous kinins [42]. There may also
be crosstalk between AT1 receptors and ACE2, because
an AT1 blocker (olmesartan) was found to increase ACE2
expression and angiotensin 17 levels in aortae from
spontaneously hypertensive rats; effects that coincided
with a reduced media-to-lumen ratio and media thickness
of the thoracic aorta [43].

A third alternative: angiotensin-converting

enzyme signalling
Although the beneficial effects of ACE inhibitors are
generally attributed to a decrease in the ACE-mediated
generation of angiotensin II and the accumulation of
bradykinin [44], a number of the effects of this class of
compounds are unrelated to changes in peptide concentrations. For example, ACE inhibitors attenuate or partly
reverse the bradykinin-induced translocation of the B2
receptor to caveolin-rich membranes and reactivate signalling events (increase in intracellular calcium ions, the
activation of extracellular-regulated kinases 1/2) in B2
receptor-desensitized cells [45]. For an ACE inhibitor to
elicit an intracellular event after binding to ACE, it was
reasoned that the enzyme should display some characteristics of a signalling molecule; for example, bind
soluble intracellular signal molecules or adaptor proteins
that provide a link to other intracellular signalling pathways. A careful investigation of the cytoplasmic tail of
ACE revealed that the intracellular cytoplasmic tail of the
enzyme is phosphorylated by the serine kinase CK2 [46],
and that the binding of an ACE inhibitor to the enzyme
elicits a biphasic increase in ACE phosphorylation [47].
ACE phosphorylation is the first reported step in the
so-called ACE signalling cascade that involves the

activation of the mitogen-activated protein kinase kinase

7 and c-Jun N-terminal kinase, as well as the enhanced
binding of the activator protein 1 transcription factor to
DNA. The ACE signalling pathway accounts for the ACE
inhibitor-induced increase in endothelial ACE and
cyclooxygenase-2 expression that occurs in vitro and
in vivo in ACE-inhibitor-treated animals [47,48]. It
remains to be determined whether or not ACE signalling
can be demonstrated in other ACE-expressing tissues.

Angiotensin-converting enzyme, angiotensin

II and diabetes
One of the most intriguing and clinically relevant findings
related to RAS inhibition using either ACE inhibitors
[49,50] or AT1 receptor antagonists [51,52] is their ability
to delay the onset of type 2 diabetes. The mechanisms
underlying this effect are unclear, but may be related
to the local RAS in the pancreas or adipose tissue
and the activation of peroxisome proliferator-activated
receptor gamma (PPARg), a nuclear receptor that influences the expression of multiple genes involved in
carbohydrate and lipid metabolism [53]. There is evidence indicating that the ACE inhibitor enalapril can
activate PPARg in aortae from apolipoprotein E-deficient
mice [54], and recent preclinical studies have indicated
that the AT1 receptor blocker telmisartan acts as a selective PPARg modulator; a property that does not appear
to be shared by all of the compounds in this class
[55,56,57]. The improved insulin sensitivity associated with ACE inhibitor and AT1 receptor blocker
therapy has also been linked to increased glucose uptake
in skeletal muscle via the enhanced synthesis and translocation of glucose transporter 4 protein to the cell surface
[58].
There are other links between ACE and diabetes; in the
diabetic kidney, ACE expression is significantly upregulated (one to threefold) in tubular epithelial cells and
infiltrating mononuclear cells [59]. Moreover, although
the blood pressure of mice with one, two, or three functional copies of the ACE gene is the same under basal
conditions, the induction of diabetes (streptozotocin)
induced a higher blood pressure and a much higher
urinary albumin excretion in mice with three ACE
genes compared with mice with one or two ACE genes
[60]. Therefore, in this model of disease, ACE levels
influence both blood pressure and disease progression.
More and more novel roles are being attributed to ACE,
and the genetically modified animals possessing three
copies of ACE have also been used to make a link
between the enzyme and the control of body weight
[61].

Conclusion
A few years ago we thought we knew all about the RAS
and much about its regulation. The identification of

12 Hormones, autacoids, neurotransmitters and growth factors

a novel ACE (ACE2) as well as an ACE-dependent

signalling pathway and intracellular angiotensin II receptors implies that the RAS still has unexpected facets
with clinical implications. The link between the RAS
and type 2 diabetes is particularly intriguing, especially in
the light of the clinical effectiveness of ACE inhibitors
and AT1 receptor blockers in delaying the onset of
diabetes.

16 Huentelman MJ, Grobe JL, Vazquez J, et al. Protection from angiotensin


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17 Donoghue M, Wakimoto H, Maguire CT, et al. Heart block, ventricular
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19 Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a
functional receptor for the SARS coronavirus. Nature 2003; 426:450454.

References and recommended reading

Papers of particular interest, published within the annual period of review, have
been highlighted as:

of special interest
 of outstanding interest
Additional references related to this topic can also be found in the Current
World Literature section in this issue (p. 84).
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Studies describing an ACE-dependent signalling pathway in endothelial cells that
is activated by the binding of an ACE inhibitor to the enzyme.
49 Lonn EM, Yusuf S, Dzavik V, et al. Effects of ramipril and vitamin E on
atherosclerosis: the study to evaluate carotid ultrasound changes in patients
treated with ramipril and vitamin E (SECURE). Circulation 2001; 103:919
925.
50 Yusuf S, Gerstein H, Hoogwerf B, et al. Ramipril and the development of
diabetes. JAMA 2001; 286:18821885.

51 Brenner BM, Cooper ME, de Zeeuw D, et al. Effects of losartan on renal and
cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N
Engl J Med 2001; 345:861869.
52 Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective effect of the
angiotensin-receptor antagonist irbesartan in patients with nephropathy
due to type 2 diabetes. N Engl J Med 2001; 345:851860.
53 Jandeleit-Dahm KA, Tikellis C, Reid CM, et al. Why blockade of the renin
 angiotensin system reduces the incidence of new-onset diabetes. J Hypertens
2005; 23:463473.
An informative review summarizing the antidiabetic actions of RAS inhibitors.
54 da Cunha V, Tham DM, Martin-McNulty B, et al. Enalapril attenuates angiotensin II-induced atherosclerosis and vascular inflammation. Atherosclerosis
2005; 178:917.
55 Benson SC, Pershadsingh HA, Ho CI, et al. Identification of telmisartan as a

unique angiotensin II receptor antagonist with selective PPARg-modulating
activity. Hypertension 2004; 43:9931002.
An interesting study that reports that some AT1 receptor antagonists act as partial
agonists and exert effects on intracellular signalling/gene expression (PPARg
activation).
56 Schupp M, Janke J, Clasen R, et al. Angiotensin type 1 receptor blockers

induce peroxisome proliferator-activated receptor-g activity. Circulation 2004;
109:20542057.
A study (published at roughly the same time as Ref. [55]) highlighting the ability of
specific AT1 receptor antagonists to activate PPARg activity in adipocytes.
57 Kurtz TW. Treating the metabolic syndrome: telmisartan as a peroxisome
proliferator-activated receptor-gamma activator. Acta Diabetologica 2005; 42
(Suppl. 1):s9s16.
58 Prasad A, Quyyumi AA. Reninangiotensin system and angiotensin

receptor blockers in the metabolic syndrome. Circulation 2004; 110:
15071512.
A well-written up-to-date review discussing the possibilities of RAS inhibition in
metabolic syndrome.
59 Das UN. Is angiotensin-II an endogenous pro-inflammatory molecule? Med

Sci Monit 2005; 11:RA155RA162.
A review focusing on the induction of cytokines, nuclear factor kappa B and
oxidative stress by angiotensin II, and discussing the anti-inflammatory actions of
RAS inhibition.
60 Huang W, Gallois Y, Bouby N, et al. Genetically increased angiotensin
I-converting enzyme level and renal complications in the diabetic mouse.
Proc Natl Acad Sci U S A 2001; 98:1333013334.
61 Heimann AS, Favarato MH, Gozzo FC, et al. ACE gene titration in mice

uncovers a new mechanism for ACE on the control of body weight. Physiol
Genomics 2005; 20:173182.
In-vivo evidence that ACE is involved in the regulation of body weight and fat
accumulation, obtained from mice with one or more copies of ACE gene.