Ren Ang

Contemporary Cardiology
C HRISTOPHER P. C ANNON , MD
SERIES EDITOR
For other titles published in this series, go to

http://www.springer.com/7677
Walmor C. DeMello Edward D. Frohlich
Editors
Renin Angiotensin System

and Cardiovascular Disease
Editors
Walmor C. DeMello Edward D. Frohlich
Department of Pharmacology Ochsner Clinic Foundation
University of Puerto Rico 1514 Jefferson Highway
P.O. Box 5067 New Orleans LA 70121
USA
San Juan PR 00936 efrohlich@ochsner.org
Medical Sciences Campus
USA
walmor.de-mello@upr.edu
ISBN 978-1-60761-185-1 e-ISBN 978-1-60761-186-8

DOI 10.1007/978-1-60761-186-8
Library of Congress Control Number: 2009933642
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Preface
Experimental and clinical evidence supports the view that the activation of the renin
angiotensin aldosterone system is involved in cardiovascular pathology including
hypertension, heart failure, myocardial ischemia, and atherosclerosis. The present
volume describes the intricacies involved in these processes, including the influence
of prorenin/renin, angiotensin II, angiotensin (1-7), and aldosterone on cardiac and
vascular functions as well as their involvement in the generation of cardiovascular
diseases. Fundamental aspects like intracellular signaling, regulation of cell volume
in the failing heart, and the presence of an intracrine renin angiotensin system are
discussed. Moreover, the role of the mineralocorticoid receptor as an important com-
ponent of the intracrine renin angiotensin system and as a regulator of extracellular
action of angiotensin II is described, reinforcing the view that aldosterone inhibitors
are helpful in the treatment of heart failure and hypertension. Let us hope the impor-
tant topics included here motivate basic and clinical investigators and contribute to
the development of new therapeutic approaches for cardiovascular diseases.
We want to thank the distinguished authors and Humana Press for the opportunity
to publish this important book.
Walmor C. DeMello
Edward Frohlich
v
Contents
1 Systemic Versus Local Renin Angiotensin Systems.

An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Walmor C. DeMello and Richard N. Re
2 Clinical Import of the Local Renin Angiotensin
Aldosterone Systems . . . . . . . . . . . . . . . . . . . . . . . . . 7
Edward D. Frohlich
3 Renin, Prorenin, and the (Pro)Renin Receptor . . . . . . . . . . . 15
Genevieve Nguyen and Aurelie Contrepas
4 Local Renin Angiotensin Systems in the Cardiovascular
System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Richard N. Re
5 Renin-Angiotensin-Aldosterone System and Pathobiology
of Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Pierre Paradis and Ernesto L. Schiffrin
6 AT1 Receptors, Angiotensin Receptor Blockade,
and Clinical Hypertensive Disease . . . . . . . . . . . . . . . . . . 59
Robert M. Carey
7 Structural and Electrophysiological Remodeling
of the Failing Heart . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Walmor C. DeMello
8 Inhibiting the Renin Angiotensin Aldosterone System in
Patients with Heart Failure and Myocardial Infarction . . . . . . 93
Marc A. Pfeffer
9 Left Ventricular Hypertrophy and Treatment with Renin
Angiotensin System Inhibition . . . . . . . . . . . . . . . . . . . . 103
Edward D. Frohlich and Javier Dez
vii
viii Contents
10 Angiotensin-(1-7), Angiotensin-Converting Enzyme 2,

and New Components of the Renin Angiotensin System . . . . . . 121
Aaron J. Trask, Jasmina Varagic, Sarfaraz Ahmad,
and Carlos M. Ferrario
11 Kinin Receptors and ACE Inhibitors: An Interrelationship . . . . 135
Ervin G. Erds, Fulong Tan, and Randal A. Skidgel
12 Kinins and Cardiovascular Disease . . . . . . . . . . . . . . . . . 151
Oscar A. Carretero, Xiao-Ping Yang, and Nour-Eddine Rhaleb
13 CMS and Type 2 Diabetes Mellitus: Bound Together by
the Renin Angiotensin Aldosterone System . . . . . . . . . . . . . 187
Deepashree Gupta, Guido Lastra, Camila Manrique,
and James R. Sowers
14 Renin Angiotensin Aldosterone System
and Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . 207
Swynghedauw Bernard, Milliez Paul, Messaoudi Smail,
Benard Ludovic, Samuel Jane-Lise, and Delcayre Claude
15 Renin Angiotensin System and Atherosclerosis . . . . . . . . . . . 215
Changping Hu and Jawahar L. Mehta
16 Renin Angiotensin System and Aging . . . . . . . . . . . . . . . . 231
Len F. Ferder
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Contributors
Sarfaraz Ahmad, MD, PhD Hypertension and Vascular Research Center,

Department of Physiology and Pharmacology, Wake Forest University School of
Medicine, Winston-Salem, NC
Robert M. Carey, MD, MACP Division of Endocrinology and Metabolism,
Department of Medicine, University of Virginia Health System, Charlottesville, VA
Oscar A. Carretero, MD Hypertension and Vascular Research Division,
Department of Medicine and Heart and Vascular Institute, Henry Ford Hospital,
Detroit, MI
Aurelie Contrepas, BS Institut de la Sant et de la Recherche Mdicale and
Collge de France, Experimental Medecine Unit, Paris, France
Claude Delcayre, PhD Centre de Recherches Cardiovasculaires INSERM
Lariboisire, PARIS, France
Walmor C. DeMello, MD, PhD Department of Pharmacology, School of
Medicine, Medical Sciences Campus, University of Puerto Rico, San Juan, PR
Javier Dez, MD, PhD rea de Ciencias Cardiovasculares, Edificio CIMA,
Pamplona, Spain
Ervin G. Erds, MD Department of Pharmacology, University of Illinois College
of Medicine, Chicago, IL
Len F. Ferder, MD Departments of Physiology, Pharmacology and Medicine,
Ponce School of Medicine, Ponce, PR
Carlos M. Ferrario, MD Hypertension and Vascular Research Center,
Edward D. Frohlich, MD Ochsner Clinic Foundation, Louisiana State University
School of Medicine, New Orleans, LA
Deepashree Gupta, MD Diabetes and Cardiovascular Center, University of
Missouri School of Medicine, and VA Medical Center, Columbia, MO
ix
x Contributors
Changping Hu, MD, PhD Division of Cardiovascular Medicine, University of

Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare
System, Little Rock, AR
Guido Lastra, MD Diabetes and Cardiovascular Center, University of Missouri
School of Medicine, and VA Medical Center, Columbia, MO
Benard Ludovic, BS Centre de Recherches Cardiovasculaires INSERM
Lariboisire, Paris, France
Camila Manrique, MD Diabetes and Cardiovascular Center, University of
Jawahar L. Mehta, MD, PhD Division of Cardiovascular Medicine, University
of Arkansas for Medical Sciences and the Central Arkansas Veterans Healthcare
System, Little Rock, AR
Genevieve Nguyen, MD, PhD Institut de la Sant et de la Recherche Mdicale,
(INSERM) and Collge de France, Experimental Medecine Unit Marcelin
Berthelot Paris, France
Pierre Paradis, MD Hypertension and Vascular Research Unit, Lady Davis
Institute for Medical Research, McGill University, Montreal. Canada
Milliez Paul, MD Centre de Recherches Cardiovasculaires INSERM Lariboisire,
Paris, France
Marc A. Pfeffer, MD, PhD Department of Medicine, Division of Cardiology,
Brigham and Womens Hospital, Dzau Professor of Medicine, Harvard Medical
School, Brigham and Womens Hospital, Boston, MA
Richard N. Re, MD Ochsner Clinic Foundation, New Orleans, LA
Nour-Eddine Rhaleb, PhD, FAHA Hypertension and Vascular Research
Division, Department of Medicine and Heart and Vascular Institute, Henry Ford
Hospital, Detroit, MI
Jane-Lise Samuel, MD, PhD Centre de Recherches Cardiovasculaires INSERM
Ernesto L. Schiffrin, MD Department of Medicine, Sir Mortimer B.
Davis-Jewish General Hospital, Hypertension and Vascular Research Unit, Lady
Davis Institute for Medical Research, McGill University, Montreal, Canada
Randhal Skidgel, PhD Department of Pharmacology, University of Illinois
College of Medicine, Chicago, IL
Messamoudi Smail, PhD Centre de Recherches Cardiovasculaires INSERM
James R. Sowers, MD Diabetes and Cardiovascular Center, University of
Contributors xi
Bernard Swyngedauw, MD, PhD Centre de Recherches Cardiovasculaires

INSERM Lariboisire, Paris, France
Fulong Tan, PhD Department of Pharmacology, University of Illinois, College of
Medicine, Chicago, IL
Aaron J. Trask, BS Hypertension and Vascular Research Center, Department of
Physiology and Pharmacology, Wake Forest University School of Medicine,
Winston-Salem, North Carolina Winston-Salem, NC
Jasmina Varagic, MD, PhD Hypertension and Vascular Research Center,
Xiao-Ping Yang, MD Hypertension and Vascular Research Division, Department
of Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, MI
Chapter 1
Systemic Versus Local Renin Angiotensin
Systems. An Overview
Walmor C. DeMello and Richard N. Re
Abstract The concept of local renin angiotensin systems in the cardiovascular

system is discussed, and evidence is presented that these systems work indepen-
dently of the systemic one.
Particular attention was given to the presence of an intracrine renin angiotensin
aldosterone system in the heart and the novel role of the mineralocorticoid receptor.
Furthermore, the influence of the renin angiotensin system on cell volume regulation
is briefly discussed.
This chapter includes an overview of these important biological concepts and
provides an introduction to the topics that are discussed in detail by different authors
throughout the book.
The renin angiotensin system (RAS) is an enzymatic cascade in which renin derived
from the juxtaglomerular cells (JG) of the kidney acts on an hepatically synthesized
substrate, angiotensinogen, to generate the decapeptide angiotensin I. This peptide
is cleaved by angiotensin-converting enzyme (ACE), primarily in the pulmonary
circulation, to the vasoconstrictor and aldosterone secretagogue, angiotensin II. The
blood pressure-elevating action of angiotensin II, together with its direct suppressive
action on JG cells and the volume expansion produced by enhanced aldosterone-
driven sodium retention, leads to the suppression of JG renin secretion, thereby
forming a negative feedback loop. Volume depletion or lowered blood pressure
stimulates renin release, leading to pressure elevation and volume retention. Ele-
vated blood pressure or hypervolemia suppresses renin release and tends to lower
blood pressure and intravascular volume. However, as powerful as this construct
is, accumulating evidence indicates that it is incomplete in that it focuses solely on
angiotensin synthesis in the circulation. For example, the blood pressure response to
ACE inhibitors, which block ACE-driven angiotensin I generation, is not predicted
by circulating renin activity, suggesting that RAS activity in tissues may be relevant
[1]. Indeed, early on it was shown that most angiotensin II generation takes place
in the arterial wall where angiotensin II is generated from RAS components taken
W.C. DeMello (B)

School of Medicine, Medical Sciences Campus, UPR, San Juan, PR USA
e-mail: walmor.de-mello@upr.edu
W.C. DeMello, E.D. Frohlich (eds.), Renin Angiotensin System and Cardiovascular 1
Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8_1,

C Humana Press, a part of Springer Science+Business Media, LLC 2009
2 W.C. DeMello and R.N. Re
up from the circulation [2]. In the follow-up to these observations, it was then noted
that components of RAS were taken up by many tissues [3], leading to the possi-
bility that angiotensin II synthesis could be locally influenced by the relative uptake
of components at those sites [4] and the synthesis of the various components can
be detected in various tissues under various conditions [3]. Together, these obser-
vations made the concept of local RASs in tissues compelling. It must be recalled
that renin in the circulation is not strictly rate-limiting for angiotensin I production.
That is, angiotensinogen circulates at a concentration close to the K m for the gen-
eration of AI by renin. Therefore, an increase in angiotensinogen will lead to an
increased production of angiotensin I [3, 5]. Thus, if a tissue were to augment tis-
sue concentration of angiotensinogen by local production, more angiotensin I and
likely angiotensin II would be generated in that tissue as compared to a tissue that
did not augment the concentration of angiotensinogen with local synthesis. Also, it
must be noted that the alteration of JG renin secretion cannot possibly equalize the
angiotensin concentrations in the two tissues, which clearly indicates local regula-
tion of local angiotensin II [5]. This, in turn, is particularly important because, of
all the components of the RAS, the synthesis of renin in tissues (with a few excep-
tions) is the most contentious. Indeed, in many tissues the reported renin secretion is
very low, suggesting that this renin could influence angiotensin production in only
a small area [4]. Nonetheless, renin upregulation has been reported in the adrenal
gland following nephrectomy where it helps maintain aldosterone secretion, as well
as in the left ventricle and other tissues such as the heart in specific circumstances
[6]. But it is clear from the arguments presented above that even in the absence of
local renin synthesis, local regulation of angiotensin production can occur through
local synthesis of other RAS cascade components [3]. Differential uptake of renin
into tissues provides another mechanism for local RAS regulation. Although in nor-
mal heart cardiac renin seems to be related to its uptake from plasma [5], evidence is
available that renin expression is increased after myocardial infarction [7] and after
stretch of the cardiomyocytes [8]. On the other hand, a renin transcript that does not
encode a secretory signal [9] and remains inside the cell is overexpressed during
myocardial infarction suggesting that intracellular renin has functional properties.
Indeed, previous studies showed that intracellular renin and Ang II administra-
tion impairs cell coupling in the heart [10, 11] and intracellular Ang II reduces the
inward calcium current in the failing heart [12], supporting the view that there is a
functional intracrine renin angiotensin system [1317]. This intracrine angiotensin
II must properly also be considered an aspect of the tissue RASs, and it may well
play an important role in such pathological processes as left ventricular hypertro-
phy, cardiac arrhythmias and cardiac myocyte apoptosis [13, 17] (see also Chapters
4 and 7).
Other studies have demonstrated upregulation of angiotensinogen and
angiotensin-converting enzyme (ACE) in tissues under normal or pathological con-
ditions. The enzyme chymase, which can substitute for ACE in the conversion of
angiotensin I to angiotesnin II, is also expressed in multiple tissues and upregu-
lated in some circumstances [17]. Even more telling is the recent demonstration
of a (pro)renin receptor in mesangial and other cells, which signals using classical
1 Systemic Versus Local Renin Angiotensin Systems 3
second messengers following the binding of prorenin or renin [18, 19]. This reveals
the hormonal nature of (pro)renin. At the same time, binding of prorenin to the
receptor activates its binding site so that the prohormone becomes enzymatically
active, generating angiotensin I in the vicinity of cell surface receptors [19]. Simi-
larly, renin bound to the receptor becomes more enzymatically active [4, 20]. These
observations make clear that the biological activity of the RAS in a tissue can be
powerfully influenced by the level of expression of the (pro)renin receptor in the
tissue a variable totally hidden from any analysis of the concentrations of circu-
lating RAS components. The potential importance of this finding is suggested by
the fact that prorenin levels are elevated in diabetic patients, and high concentra-
tions of circulating prorenin are a predictor of retinopathy a finding made all the
more compelling by the observation that prorenin can be synthesized locally in the
Mueller cells of the retina [19]. In addition, it now appears that there exist coun-
tervailing systems which while not influencing angiotensin II action at the recep-
tor nonetheless offset some of its effect. For example, an ACE homologue, ACE2,
has recently been described and studied [21]. ACE2, unlike ACE, does not con-
vert angiotensin I to angiotensin II, but rather its principal action seems to be the
conversion of angiotensin II to the hepatapeptide angiotenin (17), which operating
through its own receptor offsets many of the vasoconstrictive and growth-promoting
actions of angiotensin II [22, 23], improving impulse propagation during ischemia
reperfusion through activation of the sodium pump, reducing the incidence of slow
conduction and the generation of cardiac arrhythmias [24] (see also Chapter 10).
Recently, it was found that chronic administration of eplerenone, a mineralocorti-
coid receptor blocker, reduces the expression of AT1 receptors at surface cell mem-
brane as well as intracellularly inhibiting the intracrine and extracellular actions of
Ang II on the inward calcium current in the failing heart [25]. These findings indi-
cate that the mineralocorticoid receptor is involved in the regulation of intracellular
and extracellular actions of Ang II and lead to the concept that there is an intracrine
renin angiotensin aldosterone system (see also Chapter 7). It is possible to con-
clude that the beneficial effects of eplerenone in patients with heart failure are in
part explained by the suppression of fibrosis, hypertrophy and electrophysiological
abnormalities elicited by Ang II [26].
It is well known that regulation of cell volume is essential for normal cellular
function. Recent evidence is available that the renin angiotensin system is involved
in the regulation of heart cell volume [27] because extracellular Ang II increases
cell volume through inhibition of the sodium pump and activation of the Na-K-2Cl
cotransporter, while intracellular Ang II reduces the cell volume by activating the
Na-K pump [27]. These findings are relevant particularly to myocardial ischemia
which by itself causes cell swelling. According to these observations, the activation
of the circulating renin angiotensin system is particularly harmful during myocardial
ischemia while the activation of the intracrine renin angiotensin system might be
beneficial by decreasing the cell volume (see Chapter 7).
In conclusion, evidence is available that there are local renin angiotensin sys-
tems in the cardiovascular system, and that a functional intracrine renin angiotensin
aldosterone system contributes to cardiovascular pathology [13, 25, 28, 29].
4 W.C. DeMello and R.N. Re
References
1. Mazzolai, L., Nussberger, J., and Aubert, J.F. et al. (1998) Blood-pressure independent cardiac
hypertrophy induced by local activated renin-angiotensin system. Hypertension 31, 132430
2. Muller, D.N., and Luft, F.C. (1998) The renin angiotensin system in vessel wall. Basic Res
Cardiol 93(Supl 2), 714
3. Kurdi, M., DeMello, W.C., and Booz, G.W. (2005) Working outside the system: an update on
unconventional behavior of the renin angiotensin system components. Intern J Biochem Cell
Biol 37, 135767
4. Nguyen, G., Delarue, F., and Bu, C. et al. (2002) Pivotal role of the renin/prorenin receptor in
angiotensin II production and cellular responses to renin. J Clin Invest 109, 141727.
5. Danser, A.H.J., van Katz, J.P., and Admiraal, P.J.J. et al. (1994) Cardiac renin and
angiotensins; uptake from plasma versus in situ synthesis. Hypertension 24, 3748
6. Peters, J., Obermuller, N., Woyth, A., Peters, B., Maser-Gluth, C., Kranzlin, B., and Gretz N
(1999) Losatan and angiotensin II inhibit aldosterone production in anephric rats via different
actions on the intraadrenal renin-angiotensin system. Endocrinology 140, 67582.
7. Passier, R.C.J.J., Smits, J.F.M., Verluyten, M.J.A., and Daemen, M.J.A.P (1996) Expression
and localization of renin and angiotensinogen in rat heart after myocardial infarction. Am J
Physiol 271, H10408
8. Malhotra, R., Sadoshima, J., Broscius, F.C., and Izumo, S. (1999) Mechanical stretch and
angiotensin II differentially upregulated the renin angiotensin system in cardiac myocytes in
vitro. Circ Res 85, 13746
9. Clausmeyer, S., Reinecke, A., and Farrenkopf, R. et al. (2000) Tissue-specific expression of
a rat renin transcript lacking the coding sequence for the prefragment and its stimulation by
myocardial infarction. Endocrinology 141, 296370
10. DeMello, W.C. (1994) Is an intracellular renin angiotensin system involved in the control of
cell communication in the heart? J Cardiovasc Pharmacol 23, 6406
11. DeMello, W.C. (1995) Influence of intracellular renin on heart cell communication. Hyper-
tension 25, 11727
12. DeMello, W.C. (1998) Intracellular angiotensin II regulates the inward calcium current in
cardiac myocytes. Hypertension 32, 97682
13. DeMello, W.C., and Danser, A.J.H. (2000) Angiotensin II and the heart: on the intracrine renin
angiotrensin system. Hypertension 35, 11838
14. Re, R.N (2000) On the biological actions of intracellular angiotensin. Hypertension 35,
118990
15. Cook, J.L., Zhang, Z., and Re, R.N. (2001) In vitro evidence for an intracellular site of
angiotensin action. Circ Res 89, 113846
16. Singh, V.P., Le, B., Bhat, V.B., Baker, K.M., and Kumar, R. (2007) High-glucose-induced
regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes.
Am J Physiol Heart Circ Physiol 293(2), H93948.
17. Paul, M., Poyan, M.A., and Kreutz, R. (2006) Physiology of local renin-angiotensin systems.
Physiol Rev 86(3), 747803.
18. Nguyen, G., Burckle, C.A., and Sraer, J.D. (2004) Renin/prorenin receptor biochemistry and
functional significance. Curr Hypertens Rep 6, 12932
19. Nguyen, G., Delarue, F., Berrou, J., Rondeau, E., and Sraer, J.D. (1996) Specific receptor bind-
ing of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1
antigen. Kidney Int 50, 1897903.
20. Nguyen, G., and Danser, A.H. (2008) Prorenin and (pro)renin receptor: a review of available
data from in vitro studies and experimental models in rodents. Exp Physiol 93(5), 55763
21. Donoghue, M., Hsieh, F., and Baronas, E. et al. (2000) A novel angiotensin converting
enzyme-related carboxypeptidase(ACE2) converts angiotensin I to angiotensin (19). Circ
Res 87, E1E9.
1 Systemic Versus Local Renin Angiotensin Systems 5
22. Ferrario, C., Chappell, M., and Tallant, E.K. et al. (1997) Counterregulatory actions of
angiotensin (1-7). Hypertension 30, 53541
23. Crackower, M.A., Sarao, R., and Oudit, G.Y. et al. (2002) Angiotensin-converting enzyme 2
is an essential regulator of heart function. Nature 417, 799802.
24. DeMello, W.C. (2004) Angiotensin (1-7) re-establishes impulse conduction in cardiac muscle
during ischaemia-reperfusion. The role of the sodium pump. J Renin Angiotensin Aldosterone
Syst Dec 5(4), 2038.
25. DeMello, W.C., and Gerena, Y. (2008) Eplerenone inhibits the intracrine and extracellular
actions of angiotensin II on the inward calcium current in the failing heart. On the presence of
an intracrine renin angiotensin aldosterone system. Regul Pept 151, 5460.
26. De Mello, W.C. (2006) Beneficial effect of eplerenone on cardiac remodelling and electrical
properties of the failing heart. J Renin Angiotensin Aldosterone Syst 7(1), 406.
27. DeMello, W.C. (2008) Intracellular and extracellular renin have opposite effects on the regula-
tion of heart cell volume. Implications for myocardial ischaemia. J Renin Angiotensin Aldos-
terone Syst. 9(2), 1128.
28. Re, R.N., and Cook, J.L. (2008) The basis of intracrine physiology. J Clin Pharmacol 48,
34450.
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Chapter 2
Clinical Import of the Local Renin Angiotensin
Aldosterone Systems
Edward D. Frohlich
Abstract The concept of local renin angiotensin (and possibly aldosterone)

systems has been a relatively recent interjection to the investigative milieu. Much
interest and important studies have resulted, and reference to applicability to dis-
ease and disease mechanisms is still of innovative and imaginative clinical and
experimental studies. To this end, there are several areas of pertinence which have
evolved including the underlying causations, mechanisms, and treatment of a num-
ber of diseases. Among those fascinating and provocative study areas is the need
for additional motivated investigation related to ventricular and vascular hypertro-
phy, remodeling, and cardiac and renal failure and new thinking related to lifestyle
modifications (including those related to salt excess, obesity, and responses to vari-
ous drugs, clinically useful or otherwise). We have much confidence that these and
other areas for study will be productive and useful and will lead to important clinical
approaches and contributions on the issue of existing local RAAS.
Much of the present-day clinical and investigative considerations of the renin
angiotensin aldosterone system (RAAS) as well as this monograph concern the clas-
sically accepted endocrine RAAS system. The overall concepts involved have been
extremely important in understanding the biology, physiology, and clinical relevance
of this system as it pertains to cardiovascular and renal diseases, and they have led
to the synthesis of new classes of therapeutic agents which have changed dramati-
cally approaches to disease. Consequently, these changes have resulted in remark-
able reductions in the morbidity and mortality of cardiovascular, renal, brain, and
other diseases.
2.1 The Classical System

The framework of this classically understood system embodies the synthesis of the
enzyme renin in the kidney, the variety of mechanisms that promote and stimulate
its release by the renal juxtaglomerular apparatus, and its action on the complex
E.D. Frohlich (B)

Ochsner Clinic Foundation, New Orleans, LA
e-mail: efrohlich@ochsner.org

8 E.D. Frohlich
protein angiotensinogen which is synthesized in the liver. The consequence of this

action is the production of the decapeptide angiotensin I which, as it passes through
the pulmonary circulation, loses its terminal two peptides by virtue of the proteolytic
action of the angiotensin-converting enzyme. The resultant octapeptide angiotensin
II is the potent vasopressor agent which is responsible for vasoconstriction; release
of aldosterone by the adrenal cortex and consequent retention of sodium and water
by the kidney; stimulation of specific brain centers responsible for increased car-
diovascular adrenergic outflow and thirst; and local endothelial actions that promote
mitogenesis, hypertrophy, collagen synthesis and tissue fibrosis, apoptosis, inflam-
mation, and, no doubt, other intracellular signaling and other biological and patho-
physiological effects [1]. Already, many of these latter actions have been incorpo-
rated in our consideration of the clinical diagnosis used clinically with respect to
endothelial dysfunction [2, 3]. Although relatively recently described, there have
been several additional components to the RAAS which have intriguing biological
actions that have the potential for developing new physiological and pathological
implications [4].
2.2 The Local Systems
Although several of the foregoing actions of angiotensin II are relatively new, they
have already been inculcated into a new dimension of the classical RAAS. This then
relates to the overall concept of this monograph. It therefore concerns the concept
of local RAAS (hereafter to be considered in plurality) that affect the structure and
function of specific target organs of disease, including heart, blood vessels, kidney,
and, no doubt, other organs [5]. To this end, although to some extent considered by
some to be controversial, each of the components of these local RAAS has been
identified within these foregoing organs although certain specific components (e.g.,
the putative synthesis of the enzyme renin within the heart) of the system. Indeed,
these local systems already have important clinical and even therapeutic consid-
erations and implications in health and disease [6]. In this respect, we also have
deliberately included the hormone aldosterone in this local system since this hor-
mone has already been identified to be present in some of these systems as for vital
consideration of the existence of local RAAS [7]. Thus, although perhaps still in the
realm of speculation, consideration of these local systems and newer components
and metabolites of the system is neither premature, irrelevant, nor speculative for
present-day consideration in this monograph. This monograph has been conceived
and organized to stimulate further fundamental and clinical investigations dealing
with the impact of the RAAS in disease. Thus, the participants of this workshop are
of the unanimous opinion that these local RAAS are no longer a subject of debate;
indeed, this is an important area of fundamental and clinical study, which is the
intellectual commitment of this entire volume.
To this end, the existence of these local systems in certain organs and the infor-
mation derived from recent and current investigations provide the substance of
2 Clinical Import of the Local Renin Angiotensin Aldosterone Systems 9
tentative (but appealing) and exciting information which relates to specific clinical
problems. Thus, this rather selective and speculative discussion of local RAAS in
disease is included to tantalize the interested reader, student, and investigator in
certain specific clinical situations including the pathogenesis and pathophysiology
of ventricular hypertrophy in hypertension; ventricular and vascular remodeling in
hypertensive and ischemic cardiovascular diseases; secondary (e.g., renal) therapeu-
tic responses to disease; structural and functional responses of organs and in tox-
emias of pregnancy, to certain lifestyle and other interventions (e.g., salt excess) in
hypertension. Ever since the Framingham Heart Study demonstrated that left ven-
tricular hypertrophy (LVH) was a major risk factor predisposing the hypertensive
patient to increased morbidity and mortality associated with coronary heart disease
[8], we have been intrigued about the fundamental pathophysiological mechanisms
of LVH that account for this risk. Thus, soon after this landmark epidemiologi-
cal study, we initiated our earliest clinical and pathophysiological studies of this
problem in which we elucidated the clinical correlates associated with the develop-
ment of LVH [9]. We perceived the well-recognized concept that arterial pressure
increased as an adaptive response of the left ventricle to the progressive increase in
afterload in response to the increasing total peripheral resistance imposed by arteri-
olar constriction imposed.
Our subsequent studies introduced the feasibility of the new noninvasive tech-
nology of M-mode echocardiography in order to identify the pathophysiological
sequence in the clinical development of LVH [10]. We confirmed that coincident
with the developing increased left ventricular (LV) mass and wall thicknesses, the
earlier events associated with electrocardiographic evidence of left atrial abnormal-
ity were also identified with increased left ventricular mass and LVH. Moreover,
these structural changes were associated with functional changes of LV functional
impairment early in LVH [10]. These early findings suggested to us our ongoing
concern that the development of LVH in hypertension were not solely the con-
sequence of adaptive hypertrophy. We soon focused our attention on the func-
tional events associated with antihypertensive therapy and whether it reversed the
increased LV mass [1114]. These studies indicated that certain agents decreased
LV mass and impaired the ventricular functional responses. However, other agents
decreased LV mass and were associated with normal ventricular function follow-
ing reversal. We also showed some of those therapeutic agents that reduced LV
mass also maintained normal function when the ventricular afterload was abruptly
increased to pretreatment levels; other agents did not maintain that normal function
[1523]. These findings suggested to us that associated with treatment were intrin-
sic biological and physiological alterations which were related to the reversal of
hypertrophy and were also responsible for these disparate functional changes.
Our ensuing hypothesis was supported by our subsequent reports that the reduc-
tion of LA mass was achieved within only 3 weeks of therapy at a time when arte-
rial pressure had not been reduced. In some studies, this was achieved with doses
of some of these agents that had not even reduced arterial pressure [18, 20]. We
therefore restated our concept to the development and reversal of the increased
LV mass in hypertension, which were associated with nonhemodyanamic as well
10 E.D. Frohlich
as hemodynamic factors [24, 25]. These provocative findings permitted a further

assessment of the issue concerning whether there were additional comorbid patho-
physiological alterations associated with LVH. This concept was soon supported by
our studies in untreated naturally developing spontaneously hypertensive rats (SHR)
and their normotensive (control) Wistar-Kyoto (WKY) rats, matched for gender and
age. In these studies we learned that they developed progressive ventricular ischemia
not only in the hypertrophied LV but also in the nonhypertrophied right ventricles
and in the LV of the WKY rats. Furthermore, this progressive ischemia with aging
was closely related to increased hydroxyproline deposition and histological evi-
dence of fibrosis in the extracellular matrix of the ventricle as well as surrounding
the intramural arterioles in the chamber [26]. These findings were supported fur-
ther by additional reports demonstrating pathological changes of apoptosis [27] and
inflammatory changes [25]. Hence, we concluded that the underlying mechanisms
of risk associated with LVH in hypertension related to ischemia, fibrosis, apopto-
sis, and inflammatory changes [23, 25]. More recently, we added yet another factor
that complicates risk associated with LV certain environment factors including
excessive dietary salt-loading (vide infra) (2830].
In this chapter, I shall not discuss the important experimental and clinical evi-
dence that provides abundant clinical and experimental data demonstrating that
angiotensin II contributes importantly to the development of LVH as well as the
remodeling of the LV and the arterioles in clinical and experimental hypertension.
This is the subject of separate chapters in this monograph [31, 32]. The evidence
is abundant with reference to the numerous well-designed placebo-controlled multi-
center pharmacological clinical trials involving administration of either angiotensin-
converting enzyme agents or angiotensin II type 1 receptor blocking agents to
patients following myocardial infarction. These trials demonstrated the efficacy of
these drugs in reducing not only arterial pressure but cardiovascular morbidity and
mortality, cardiac failure, and even a second myocardial infarction [6].
2.3 Structural and Function Response of Organs to Salt-Loading
Abundant clinical and experimental evidence has accumulated in recent years to

the response of various organs (i.e., heart, vessels, kidney) to excessive salt-loading
[2830, 33). Until relatively recently, much evidence of risk with salt-loading has
been ascribed to increase in arterial pressure; however, more recent reports have
demonstrated clearly that salt-loading (experimentally as well as clinically) was
associated with increased cardiovascular morbidity and mortality as well as struc-
tural and functional alterations of heart, aorta, and kidney [3436). Recent data have
shown that co-treatment with angiotensin II receptor antagonists or angiotensin-
converting enzyme inhibitors along with the salt-loading will prevent the struc-
tural and functional end-organ damage [30, 33, 35]. The reader is referred to those
specific references that provide abundant data and references to support the fore-
going statements. Moreover, sodium-restricted diets in prehypertension patients
will significantly reduce cardiovascular morbidity and mortality as compared with
control group patients whose daily sodium diet was not reduced [36]. These findings
provide important data that relates the data derived from chronic salt-loading diets
in the earlier epidemiological studies that demonstrated a close relationship between
salt-loading and the prevalence of hypertension in large population groups [3739].
2.4 Secondary Organ Responses of Therapy

to Certain Treatment
Over the past five or more decades of antihypertensive therapy and the well-
documented evidence of associated reduction in cardiovascular morbidity and mor-
tality, a disturbing conundrum has complicated this therapeutic effort [33]. Thus,
each national and international report has attested to the remarkable reduction in
morbidity and mortality of such disease endpoints in hypertensive emergencies,
stroke, and coronary heart disease [40, 41]. However, over the years, the successive
publications of these very same reports have continued to provide an ever-increasing
prevalence, morbidity and mortality resulting from end-stage renal disease and of
cardiac failure [40, 41]. How can we reconcile these startling data? In response to
this shocking and as yet unresolved conundrum, we have suggested that this may be
the result of long-term stimulation or ineffective inhibition of the local cardiac and
renal renin angiotensin systems. Indeed, there are abundant experimental data which
have demonstrated that prolonged diuretic treatment promotes structural and func-
tional renal abnormalities which can be prevented by co-existent treatment with an
angiotensin-converting enzyme agent or an angiotensin II type 1 angiotensin recep-
tor blocker [42, 43]. This led to our suggestion resulting from long-term diuretic
therapy, there is a secondary increase of renin generation in the kidney that promotes
the local synthesis of angiotensin II and its attendant pathophysiological alterations
from secondary renal renin generation [34]. These latter studies have demonstrated
that in addition to promoting renin release from the juxtaglomerular apparatus of the
kidney, a second source of renin production occurs in renal tubular cells [44, 45]. In
addition, salt-loading without adequate treatment with either an ACE inhibitor or an
angiotensin II type 1 receptor blocker may not protect or prevent stimulation of the
local cardiac RAAS. These salt/pharmacological stimuli or inhibition of local renal
and cardiac RAAS may be analogous to the multiplicity of Yin/Yang biological sys-
tems in the body. Therefore, unless the consequent events stimulating the increased
renin synthesis and angiotensin II generation are prevented, the adverse structural
and functional cardiac and renal biological events may result.
2.5 Toxemias of Pregnancy
Finally, a word or two may be in order concerning yet another clinical expression
of pathological stimulation of a local RAAS in the uterus or other female genital
organs. Several recent reports have suggested that the utero-placental unit may be
the source of stimulated synthesis of components of the RAAS [46, 47]. In part, this
12 E.D. Frohlich
may be related to inadequate perfusion of the utero-placental unit and/or a conse-

quent relative hypoxemia stimulation, endothelial dysfunction of that unit, upreg-
ulation of specific genes, generation of autoantibodies, and generation of certain
humoral or hormonal factors, inflammatory changes and production of an increased
arterial pressure and proteinuria that are characteristic of pre-eclampsia or eclamp-
sia [48, 49]. Each of these possible pathophysiological changes may be responsible
for the establishment of toxemia alone or in association with preexisting or other-
wise predisposed underlying mechanisms of hypertensive disease. Although these
provocative findings are of great significance, what is most important is that this
much neglected area for study has now captured much needed interest and work.
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Chapter 3
Renin, Prorenin, and the (Pro)Renin Receptor
Genevieve Nguyen and Aurelie Contrepas
Abstract The discovery of a receptor for renin and for its inactive precursor
prorenin, and the introduction of renin inhibitors in therapeutic, has renewed the
interest for the physiology of the renin angiotensin system (RAS) and has brought
prorenin back in the spotlight. The receptor known as renin for (Pro)Renin Receptor
binds both renin and prorenin, and binding triggers intracellular signaling involving
the MAP kinases ERK1/2 and p38. The MAP kinases activation in turn upregu-
lates the expression of profibrotic genes, potentially leading to fibrosis, growth, and
remodeling. Simultaneously, binding of renin to (P)RR increases its angiotensin
I-generating activity, whereas binding of prorenin induces the inactive prorenin to
become enzymatically active. These biochemical characteristics of (pro)renin bind-
ing to (P)RR allow to distinguish two aspects for the new (pro)renin/(P)RR sys-
tem, an angiotensin-independent function related to the intracellular signaling and
its downstream effects and an angiotensin-dependent aspect related to the increased
generation of angiotensin I on the cell surface. Ongoing experimental studies should
now determine which of the two aspects is the most important in pathological
situations.
List of Abbreviations
(pro)renin: designate renin and prorenin

AOG: angiotensinogen
Ang I and Ang II: angiotensin I and angiotensin II
ACE: angiotensin-converting enzyme
HRP: handle region peptide
(P)RRB: (pro)renin receptor blocker
G. Nguyen (B)
Institut de la Sant et de la Recherche Mdicale, Pars, France
e-mail: genevieve.nguyen@college-de-france.fr

16 G. Nguyen and A. Contrepas
3.1 Introduction
The discovery of a specific receptor for renin and for its precursor, prorenin, has
modified our conception of renin being just an enzyme responsible for the cleav-
age of angiotensinogen and of prorenin being just an inactive proenzyme. The
receptor named (P)RR binds with similar affinity to renin and prorenin. Binding to
the receptor allows these enzymes to display increased enzymatic activity on the
cell surface and trigger intracellular signaling that in turn modifies gene expression.
This implies that renin may also be considered as a hormone and that a function was
finally found for prorenin. Information on the role of the (P)RR in organ damage
was obtained only recently, and experimental models suggest that (P)RR may play a
role in the development of high blood pressure and of glomerulosclerosis, in cardiac
fibrosis, in diabetic nephropathy and retinopathy by non-proteolytically activating
prorenin. Importantly, blocking prorenin/(P)RR interaction with a putative (P)RR
blocker called handle region peptide (HRP) was claimed to not only prevent dia-
betic nephropathy but also reverse the glomerulosclerosis of diabetic nephropathy.
If this is true, then it would make (P)RR a major therapeutic goal.
3.2 Renin and Prorenin
The term renin is used to cover two entities:
renin, the mature enzyme which is catalytically active in solution and

prorenin, the proenzyme form of renin which is virtually inactive in solution.
Prorenin is synthesized in many organs, the kidney of course, and also the eye,
the brain, the adrenal gland, the submandibular gland, the glands of the reproductive
system and the adipose tissue. All these tissues are able to secrete inactive prorenin
in the surrounding milieu and in plasma, but the only tissue able to mature and
secrete active renin is the kidney. Indeed, prorenin, but not renin, is still detectable in
blood after bilateral nephrectomy, although prorenin levels are lower than in normal
subjects indicating that, although kidney is the main if not the only source of renin
in the body, other tissues are able to release prorenin in the circulation [1, 2].
3.2.1 Renin
Renin is an aspartyl protease with a typical structure made of two lobes. The cleft
in between the lobes contains the active site characterized by two catalytic aspar-
tic residues. Renin is a highly specific enzyme and has only one known substrate,
angiotensinogen (AOG). Renin cleaves AOG to generate angiotensin (Ang) I that
is converted into Ang II by the angiotensin-converting enzyme. In addition to its
substrate specificity, renin catalytic activity is species-specific and renin can only
3 Renin, Prorenin, and the (Pro)Renin Receptor 17
cleave AOG of the same species. Renin is synthesized by the renin-producing cells
of the juxtaglomerular apparatus (JGA) and is stored as active enzyme in secretory
granules from which it is released upon acute stimulation of the JGA. Renin has
also been called active renin in opposition to the enzymatically inactive form of
renin, prorenin [3].
3.2.2 Prorenin
Being the precursor of renin, prorenin was assumed to have no function of its own,
and yet it represents 7090% of total renin in human plasma. The absence of enzy-
matic activity of prorenin is due to the fact that a 43-amino acid N-terminal pros-
egment covers the cleft of the active site. Unlike for the proenzymes of trypsin
and of cathepsin D, prorenin does not undergo auto-activation in the plasma ant its
activation takes place under two circumstances: a proteolytic activation by a pro-
convertase which identity is still not established and that removes the prosegment,
an irreversible process that occurs in physiology in the renin- producing cells of the
juxtaglomerular apparatus exclusively; and non-proteolytic activation in a test tube
by exposure to low pH (pH < 3.0) or cold (4 C) and which can be imagined as a
reversible unfolding of the prosegment. In plasma and in physiological conditions,
however, approximately 2% of prorenin is in the open, active form and can display
enzymatic activity, whereas 98% is in closed and inactive form [3].
In contrast to renin, prorenin is released constitutively and renin and prorenin
levels are usually well correlated. However, under some physiopathological cir-
cumstances such as pregnancy and diabetes, prorenin levels exceed by far those
of renin. In diabetes mellitus complicated by retinopathy and nephropathy, prorenin
is increased out of proportion to renin and this increase starts before the occur-
rence of microalbuminuria, so that prorenin level was suggested to be a marker of
microvascular complications in diabetic patients [4, 5]. Pregnant women also have
high plasma prorenin levels, likely derived from the ovaries. The reason for the ele-
vated prorenin levels in diabetes and pregnancy is unknown.
3.3 The (Pro)Renin Receptor

Interestingly, the renal vasodilator response to captopril in diabetic subjects corre-
lated better with plasma prorenin than plasma renin [6]. Possibly therefore, prorenin
rather than renin is responsible for tissue angiotensin generation despite the absence
of proreninrenin conversion that cannot occur elsewhere than in the JGA cells [7].
In support of this concept, transgenic rodents with inducible prorenin expression
in the liver display increased cardiac Ang I levels, cardiac hypertrophy, hyper-
tension, and/or vascular damage without evidence for increased circulating renin
or angiotensin [810]. Even more surprising, increased tissue Ang I formation
occurred even when expressing a non-activatable prorenin variant mutated in the
site of cleavage of the prosegment [11]. Therefore, it seems logical to assume that
prorenin accumulates in tissues, e.g., via a receptor-dependent mechanism, where it
can be activated in a non-proteolytic manner.
Several proteins able to bind renin and prorenin have been described, an intracel-
lular renin-binding protein (RnBP) [12] and the mannose 6-phosphate/insulin-like
growth factor II receptor (M6P/IGF2R) [1315]. The intracellular RnBP was found
to be an inhibitor of renin activity and its deletion affected neither blood pressure
nor plasma renin [16], and it is now believed that the M6P/IGF2R is a clearance
receptor for renin/prorenin [17]. This leaves the (Pro)Renin Receptor [(P)RR] as the
most promising candidate for the tissue uptake of circulating renin/prorenin.
3.3.1 Biochemistry of the (P)RR
The (pro)renin receptor is a 350-amino acid receptor with a single transmembrane

domain, like receptors for growth factors [18]. There is no homology with any
known protein based on the nucleotide and the amino acid sequence of (P)RR.
Homologies in the tertiary structure have not yet been determined due to the lack of
knowledge on the crystal structure of (P)RR. The receptor binds both renin and
prorenin, with affinities in the nanomolar range, and the encoding gene, called
ATP6AP2 (see below), is located on the X chromosome in locus p11.4.
The initial characteristics of the (P)RR were:
1. Renin and prorenin bound to the receptor are not internalized or degraded but
remain on the cell surface.
2. Renin bound to the receptor displays increased catalytic activity as compared to
renin in solution.
3. Receptor-bound prorenin displays Ang I-generating activity in the absence of
cleavage of the prosegment, most likely due a conformational change induced
by binding and non-proteolytic activation of prorenin.
4. (Pro)renin binding triggers intracellular signalization involving the mitogen-
activated protein (MAP) kinase ERK1/2 and p38.
Further studies confirmed ERK1/2 phosphorylation and showed that it was due to
MEK phosphorylation and provoked Elk phosphorylation [1922]. Moreover, ERK
1/2 activation resulted in the upregulation of transforming growth factor 1 gene
expression, the subsequent upregulation of genes coding for profibrotic molecules
such as plasminogen-activator inhibitor-1, fibronectin, and collagens, and the induc-
tion of mesangial cell proliferation [19, 20, 23]. The ERK1/2 pathway is not the only
signaling pathway linked to the (P)RR as the receptor also appears to activate the
MAP kinase p38-heat shock protein 27 cascade [24] and the PI3K-p85 pathway
[25]. Importantly, the latter results in the nuclear translocation of the promyelocytic
zinc finger transcription factor, which downregulates the expression of the (P)RR
itself [25, 26]. In other words, high (pro)renin levels will suppress (P)RR expression,
thereby preventing excessive receptor activation.
Prorenin binding (Fig. 3.1) and its subsequent non-proteolytic activation was
confirmed both in primary cells [27] and in cells with transient overexpression
of (P)RR [28]. Data in rat aortic vascular smooth muscle cells overexpressing the
human (P)RR suggested that prorenin binds with higher affinity to the receptor than
renin, so that in vivo prorenin might be the endogenous agonist of the receptor [27].
The fact that both prorenin and renin are capable of binding to the (P)RR implies that
the domains involved in the interaction between (P)RR and the (pro)renin molecule
are different from the active site and are not restricted to the prosegment of prorenin.
Unfortunately, due to the difficulties in generating purified recombinant (P)RR, no
structurefunction studies are currently available, which would allow the identifica-
tion of the domains of the (P)RR and (pro)renin involved in binding. In the absence
of such structurefunction studies or of an X-ray crystallographic structure of the
(P)RR, it is difficult to design antagonists for the (P)RR. Nevertheless, Suzuki et
al. [29] made the interesting observation that, when bound to prorenin, an anti-
body against the sequence I11P FLKR15P of the prosegment was able to open the
pro-fragment, yielding a non-proteolytically activated prorenin in a manner sim-
ilar to the putative mechanism of (P)RR binding-induced prorenin activation. They
named this region of the prosegment the handle region. Based on this observation,
Ichihara et al. [30] tested a 10 amino acid peptide encompassing the handle region
and called HRP for handle region peptide as a blocker of prorenin-(P)RR binding. In
diabetic rodents, they reasoned that diabetes would increase prorenin synthesis, thus
Fig. 3.1 Schematic representation of the angiotensin II-dependent and independent consequences
of (pro)renin binding to (P)RR and of (P)RR activation. Adapted from [47]
creating optimal conditions to test the efficacy of HRP in vivo. Indeed, HRP could
totally prevent or even reverse diabetic nephropathy [30, 31] and blocked ischemia-
induced retinal neovascularization and ocular inflammation in endotoxin-induced
uveitis [32]. Moreover, it diminished cardiac fibrosis in stroke-prone spontaneously
hypertensive rats [31]. Taken together, these data strongly suggest that the prorenin-
(P)RR axis plays an essential role in end-organ damage in diabetic and inflammatory
pathologies. HRP was subsequently renamed a (P)RR blocker.
However, in vitro and in vivo studies by others did not reproduce the protec-
tive effect of HRP on organ damages well as they did not support the inhibition
of prorenin binding to its receptor by HRP [22, 27, 33]. Even more surprising, an
FITC-labeled HRP also bound to cells devoid of the (P)RR on the plasma membrane
[22]. If there is no demonstration that HRP can really block (pro)renin binding to
the (P)RR, thus one may wonder why it is so successful if not blocking renin
(P)RR interaction. At this moment, it cannot be ruled out that HRP also exerts other
non-(P)RR related effects, particularly in diabetic animals. Clearly, more work is
needed to unravel its mechanism of action before HRP can truly be called a (P)RR
blocker.
3.3.2 (P)RR in Experimental Models of Cardiovascular

and Renal Diseases
The high blood pressure occurring in a transgenic rat model targeting human (P)RR
expression to vascular smooth muscle cells suggests a pathological role of the
(P)RR in raising blood pressure [34]. Ubiquitous over-expression of the human
(P)RR resulted in proteinuria and glomerulosclerosis [35] and in cyclooxygenase-2
upregulation [36]. Both targeted and ubiquitous (P)RR expression left the plasma
levels of renin and angiotensin unaltered, but did cause a rise in plasma aldosterone.
Finally, in a Goldblatt model of hypertension, a parallel increases in (P)RR and
renin was suggested to be profibrotic in the clipped kidney [37] and an increase of
(P)RR expression was described in diabetic rats [38]. Although the claimed benefi-
cial effects of HRP in diabetic rodents and stroke-prone spontaneously hypertensive
rats are suggestive for a role of the (P)RR in fibrosis and glomerulosclerosis, no
increased (P)RR expression was described in these models [30, 31, 39]. In addition,
it should be noted that glomerulosclerosis did not occur in transgenic ren-2 rats
with inducible prorenin expression [10], despite the fact that such rats, following
induction, displayed 200-fold higher prorenin levels, with no change in renin. This
argues against the concept that prorenin, through a direct interaction with its recep-
tor, induces glomerulosclerosis. Of the two means classically used to establish the
role of a receptor in pathology, the antagonist, HRP, is still speculative and the total
knock-out of the (P)RR is, surprisingly for a component of the rennin angiotensin
system (RAS), not possible [40]. Therefore, the generation of (P)RR conditional
knock-out mice is becoming mandatory and such animals will allow to further estab-
lish the role of (P)RR in disease.
3.3.3 Unexpected Properties and Ontogeny of the (P)RR

There is only one gene called ATP6ap2 coding for the full-length protein known as
(P)RR. All other truncated forms of (P)RR derive from intracellular processing of
the full-length form. The reason why the (P)RR gene is called ATP6ap2 was because
a truncated form of the (P)RR, composed of the transmembrane and cytoplasmic
domains of (P)RR, had been co-purified with a vacuolar H+ -ATPase (V-ATPase)
[41]. This V-ATPase is a complex, 13-subunit protein, essential to maintain an acidic
pH in intracellular vesicles and to regulate cellular pH homeostasis [42], but (P)RR
is not a subunit of this V-ATPase.
The necessity of an intact (P)RR/ATP6ap2 gene in early development is stressed
by the observations that in zebra fish, the mutation of (P)RR/ATP6ap2 gene pro-
voked the death of the fish before the end of the embryogenesis [43] and that in
rodents (P)RR/ATP6ap2 gene expression is ubiquitous and early in development
[44]. Whereas renin expression can be detected in large intrarenal arteries only at
15.5 days of gestation, (P)RR mRNA is already present on day 12 in the ureteric
bud and at later stages in vesicles and S-shaped bodies (Fig. 3.2). In newborn mice
(P)RR expression is high in epithelial cells of distal, proximal, and collecting tubules
and low in glomeruli and arteries [44]. These observations in zebra fish and in the
developing kidney suggest that the (P)RR has functions essential for cell survival
and proliferation that are unrelated to the RAS.
Fig. 3.2 In situ hybridization with 35 S-labelled mouse (P)RR (left) and mouse renin (right)
riboprobes
Analysis of the sequence of (P)RR-coding cDNA shows that sequence coding

for the transmembrane and the intracellular domain putatively associated with the
V-ATPase is remarkably conserved between invertebrates and vertebrates, whereas
the cDNA sequence coding for the extracellular domain responsible for renin and
prorenin binding is conserved in vertebrates only [45]. This leads to the postulate
that the (P)RR/ATP6ap2 gene may result from the fusion of two genes, an ancient
gene (corresponding with the C-terminus) coding for a protein essential for cell sur-
vival and a more recent gene in vertebrates (corresponding with the N-terminus)
which binds renin and prorenin [40]. However, to date, we have no arguments to
confirm or to infirm that the (P)RR role in cell survival is related to V-ATPase
activity.
3.4 Conclusion
The discovery of the (P)RR has confirmed the hypothesis of Tigerstedt and Bergman
more than a century ago that renin is a hormone [46]. Now, the (P)RR also endows
prorenin with a function that was suspected over 25 years ago by Luetscher and Wil-
son in diabetic patients (1985). Experimental studies suggest that the (P)RR might
be a major target in cardiovascular disease and in diabetes-induced organ damage,
and tissue-specific knock-out of (P)RR should soon establish whether the (P)RR
plays a role in cardiovascular pathologies and in diabetes and to what degree HRP
exerts (P)RR-dependent effects.
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Chapter 4
Local Renin Angiotensin Systems
in the Cardiovascular System
Richard N. Re
Abstract The renin angiotensin system (RAS) is an established regulator of

intravascular volume and arterial pressure. It is now clear that complete and par-
tial RASs exist in multiple tissues, including the cardiovascular system, with the
result that local regulation of angiotensin can occur. In addition, newly identified
factors such as ACE 2 and the (pro)renin receptor expand the potential physiolog-
ical actions of these tissue RASs. Here evidence for the existence and functional
relevance of local RASs in cardiovascular tissues is reviewed.
4.1 Introduction
There is abundant evidence to indicate that components of the renin angiotensin sys-
tem (RAS) are found in, and act in, cardiovascular tissues. In some cases evidence
strongly points to local synthesis of these moieties, while in others uptake from
the circulation predominates [15]. However, in either case, there exists the oppor-
tunity for local regulation of angiotensin production in various tissues. Moreover,
tissue-level regulation also occurs via the actions of newly discovered angiotensin-
converting enzyme homologue (ACE-2), which cleaves angiotensin II to produce
angiotensin [17], a peptide with actions that in many instances counteract those
of angiotensin II itself [6, 7]. Receptor interactions and modulation can affect the
net action of RAS peptides in tissues. For example, heterodimerization of the AT-
1 angiotensin II receptor with the bradykinin receptor leads to enhanced activity
of the AT-1 receptor, and this may play a role in the angiotensin II sensitivity
seen in patients with pre-eclampsia [8, 9]. Also the AT-1 receptor can transacti-
vate the epidermal growth factor (EGF) receptor at the cell surface and thereby
enhance cell growth [10]. A reciprocal interaction between the AT-1 receptor and the
lectin-like oxidized low-density lipoprotein (LDL) receptor results in upregulated
R.N. Re (B)
Ochsner Clinic Foundation, New Orleans, LA
e-mail: rre@ochsner.org

26 R.N. Re
angiotensin signalling in the environment of high-circulating oxidized LDL [11]. In

addition, two receptors for (pro)renin have recently been described: one an appar-
ent clearance receptor and the other having the capacity to enhance angiotensinogen
cleavage at the cell membrane, leading to enhanced generation of angiotensin II in
the near vicinity of the AT-1 receptor [1214]. Moreover, the binding of prorenin
to this receptor results in the activation of prorenin such that angiotensinogen can
be cleaved and angiotensin II subsequently produced at the target cells. This latter
receptor is widely expressed and in particular is found on coronary artery smooth
muscle cells and on mesangial cells [1214]. The physiological relevance of these
findings is suggested by the recognition that elevated circulating prorenin levels are
found in diabetics, and although this circulating prorenin was heretofore thought to
be inert, circulating prorenin concentrations correlate with the extent of microvas-
cular complications in these patients [15]. The marked expression of prorenin in
the diabetic eye also suggests a role for prorenin activation at its receptor in human
disease [16].
Thus, the actions of local RAS systems in cardiovascular tissues are complex and
in some cases remain controversial. Early on, there was considerable debate over
whether or not renin was synthesized in cardiovascular tissues [17]. Although the
great majority of renin in these tissues is taken up from the circulation, there are per-
sistent reports that renin can be synthesized in cardiovascular tissues of experimental
animals and in other tissues [2, 18]. But irrespective of the degree of local synthesis
in the cardiovascular tissues of man or experimental models, it must be recognized
that the availability of angiotensinogen and ACE in tissues can nonetheless lead to
dramatic differences in tissue angiotensin II production in different tissues that
is, tissue angiotensin II production can be locally regulated [3]. Differential uptake
of these components could produce this kind of local regulation. In addition, there
is evidence for the synthesis of ACE and angiotensinogen in various tissues (for
example, the left ventricle in the case of angiotensinogen and the vessel wall in
the case of ACE) under specific physiological conditions. These findings are not as
controversial as is the issue of tissue renin synthesis; local synthesis of ACE and
angiotensinogen therefore provides yet another means of local tissue RAS regula-
tion in the cardiovascular system [3, 18, 19]. Moreover, there is good evidence for
the synthesis of chymase, a second enzyme capable of converting angiotensin I to
angiotensin II in heart and other tissues depending on species [20]. It must also be
noted that renin gene expression resulting in prorenin synthesis occurs in multiple
sites [15, 2127]. Thus, even in the absence of conversion to active renin, tissue-
derived prorenin could generate angiotensin II independent of circulating renal renin
after prorenin binding to its receptor in tissue or it could function by direct receptor
stimulation [26, 28].
In order to approach this complex subject, the evidence for local cardiovascu-
lar RAS regulation in man will first be discussed. Thereafter, the more extensive
literature on local cardiovascular systems in selected experimental models will be
reviewed both to point the way towards new lines of experimentation and to suggest
novel physiologic roles of locals RASs in human physiology.
4 Local Renin Angiotensin Systems in the Cardiovascular System 27
4.2 Studies in Man

The most direct studies of the cardiovascular RAS in man have been conducted
by Sernieri and colleagues. Initially, they studied normal volunteers on both low-
and high-salt diets and determined the angiotensin II gradient across the heart [29].
Depending on conditions, the hearts of these volunteers either extracted or released
angiotensin while transiting the heart. Surprisingly, cardiac synthesis of angiotensin
I and angiotensin II was lowest when the patients were on a low-sodium diet (that
is, when circulating renin and angiotensin II were high) and greatest on the high-
sodium diet (when circulating renin and angiotensin were low). Thus, this study pro-
vided clear evidence of locally regulated angiotensin II production. Later, this group
took advantage of the fact that some patients suffering from severe heart failure
undergo cardiac transplantation, meaning that both diseased and normal myocardia
can easily be biopsied for study [30]. Hearts removed from patients suffering from
cardiomyopathy reveal increased tissue expression of angiotensinogen, ACE and
chymase by PCR and in situ hybridization as compared to normal donor hearts. No
renin signal was detected in the tissues, however, and presumably the local upregu-
lation of angiotensinogen and ACE allowed renin to be taken up from the circulation
to drive local synthesis of angiotensin II. Also, the gradients of angiotensin I and II
across the heart were elevated in patients with congestive heart failure and were cor-
related with wall stress to a greater degree than to circulating renin activity. Once
again, this study points to a role for locally generated angiotensin in the heart and,
taken with studies in diabetic patients, suggests that increased tissue concentrations
of angiotensin II are associated with apoptosis, a role in cardiac pathobiology [2].
An area of some controversy is the possibility that cardiac tissue can synthesize
aldosterone from circulating steroids or even directly from cholesterol. Catheteri-
zation studies revealed a gradient of aldosterone, suggesting cardiac production in
patients with congestive heart failure or hypertension [3133]. These studies, how-
ever, do not necessarily imply aldosterone synthesis in the heart but could alterna-
tively reflect differential uptake and release. This, coupled with ongoing controversy
regarding the possibility that cardiac tissue can synthesize aldosterone, has led to
uncertainty regarding the physiologic significance of these results. But the issue of
local synthesis aside and therefore the related issue of whether the local cardiac
RAS regulates aldosterone levels so as to form a local renin angiotensin aldosterone
system the variability of aldosterone release from the heart and its association
with disease suggests that local regulation of cardiac aldosterone concentration by
one mechanism or another does in fact occur.
Additional support for local regulation of angiotensin in cardiovascular tissues
comes from studies of diabetic patients. In this group plasma renin activity is low; it
had been conjectured that the diabetic kidney was not capable of activating prorenin
adequately. However, when renovascular resistance was determined in these patients
before and after ACE inhibition, AT-1 blockade, or renin inhibition, it was found that
renovascular resistance responded briskly to all three agents [3436]. Also, treat-
ment with ACE inhibitors resulted in marked increases in circulating plasma ren-
nin activity [34]. Collectively, these results suggest the likelihood that renal tissue
28 R.N. Re
production of angiotensin II results in increased renovascular resistance and renin

suppression. The fact that all three agents reverse the effect on resistance indicates
that any tissue source of angiotensin II is renin driven. In fact, the response to the
renin inhibitor aliskiren is greater than that to the other agents, raising the possi-
bility that prorenin activation at the (pro)renin receptor a site in close proximity
to the AT-1 receptor could account for this differential sensitivity to aliskiren,
which could block the action of prorenin directly and therefore effectively blunt the
RAS cascade [36]. These findings must be seen in the context of animal studies
demonstrating synthesis of renin in the renal tubules with subsequent generation
of angiotensin II from locally produced and filtered angiotensinogen. It is notewor-
thy that angiotensin II infusion actually upregulates the local production of renin
and angiotensinogen, indicating a local renal tubular RAS [37, 38]. To the extent
that angiotensin II so produced can migrate to the juxtaglomerular cells to suppress
renin release and can traffic to the renal vasculature to cause vasoconstriction, this
system could explain the findings described above in diabetic subjects.
4.3 Studies in Animal Models
Compared to the relative paucity of direct data related to the cardiovascular RAS in
man, there is a large body of work studying this system in animals. Interpretation of
this work is made complex by virtue of known and unknown species differences in
the RAS. For example, the mouse has a second renin gene (REN 2) expressed pri-
marily in salivary glands and producing a nonglycosylated renin moiety [39]. The rat
expresses two related but distinct AT-1 receptors [40]. This divergence in the RAS
among species carries over to the local systems and their functioning; this demands
caution in extrapolating animal studies to man. An additional caveat derives from
the fact that many cell culture studies of the local RAS involve neonatal cells, which
are more easily cultured than adult cardiomyocytes. Here too, caution must be exer-
cised in extrapolating results to adult animals.
That said, there is good evidence for the regulated synthesis of various RAS
components in the cardiovascular and other tissues of experimental animals. Here
local RAS activity in the heart, adrenal gland and kidney will be discussed.
4.3.1 Cardiac RAS
More than 25 years ago, renin gene expression was first reported in the rodent heart,
and this observation has been repeated in various species over the ensuing decades
[2, 18, 41]. To be sure, expression is low and in some systems is only seen in patho-
logical states. For example, rapid cardiac pacing leads to the upregulation of renin
gene expression in the dog heart [42]. In rats myocardial infarction is associated with
the upregulation of renin expression in monocytes and myofibroblasts in the infarc-
tion zone [18]. At the same time, cardiac tissue angiotensin I and II levels fall to
very low levels following nephrectomy, indicating the primacy of circulating renin
in the generation of angiotensin in the cardiac interstitium. This has led some to the
dogmatic view that all cardiac renin is derived from the circulation [17]. This view
assumes that the entire physiological relevance of tissue renin lies in the generation
of interstitial angiotensin. This need not be the case. Recently, a second renin tran-
script, encoding a renin lacking the signal sequence for secretion, has been described
by three groups. In the rodent adrenal gland both genes are expressed, while in the
juxtaglomerular cells only the secreted form is found. However, in the heart only
the nonsecreted form is expressed, and this expression is markedly upregulated by
myocardial infarction [2123]. This intracellular renin may be pathogenic (see Intra-
cellular RASs).
Irrespective of the role of locally produced renin in the heart, there is clear
evidence for renin uptake from the circulation along with local synthesis of
angiotensinogen, leading to locally regulated angiotensin I production followed by
its conversion to angiotensin II by ACE or chymase. The upregulation of angiotensin
production in the ventricles of transgenic animals has been shown to lead to left ven-
tricular hypertrophy, and so this local system is likely relevant to myocardial stress
responses and pathology [43]. The synthesis of aldosterone in the hearts of experi-
mental animals has been reported, but this remains controversial.
4.3.2 Adrenal RAS

There is a consistent body of evidence to indicate the synthesis of renin by the rodent
adrenal gland. Indeed, following nephrectomy adrenal renin synthesis increases in
these animals to the extent that circulating renin levels remain detectable as the
result of spillover into the circulation [44]. Moreover, this adrenal renin appears
to be biologically important in so far as it maintains adrenal aldosterone synthesis
in the absence of renal renin [22, 24, 25, 45, 46]. It is also of note that following
nephrectomy renin accumulates in crystalline structures in adrenal mitochondria, the
sites of aldosterone synthesis. Thus, it can be conjectured that a local intracellular
RAS exists in these cells. Indeed, several groups have reported the existence of a
renin variant termed renin exon1A, which lacks the signal sequence for secretion
and is predicted to encode a nonsecreted but active renin (as opposed to prorenin),
which could act within the cell without the necessity of proteolytic activation [21
23]. Studies indicate that the renin that accumulates in the mitochondria of anephric
animals is this nonsecreted form of renin and is active in aldosterone regulation
[22, 24, 25].
4.3.3 Renal RAS
As noted earlier, there is good evidence for an autonomous tubular renin angiotensin
system that is upregulated in high angiotensin II states, including renal artery
30 R.N. Re
stenosis. This local system appears to play a role in renal autoregulation and may
spill over to influence juxtaglomerular renin secretion and renovascular resistance
[37, 38]. At the same time there is evidence to suggest that direct (pro)renin effects at
mesangial and possibly other kidney cells are physiologically important. It has been
reported that a peptide designed to mimic the handle region of prorenin, and there-
fore to block the binding of prorenin to the prorenin receptor, can blunt the glomeru-
lar pathology in a rodent model of diabetes. It did this in an angiotensin-independent
manner as evidenced by a beneficial effect in animals lacking the AT-1 receptor
[47]. These results suggest a novel approach to the prevention of glomerulosclero-
sis. However, there exists controversy about these findings because the observations
have not yet been successfully repeated. Also, it is unclear why a peptide directed
against prorenin binding would be so effective given the fact it is expected to have
no effect on renin binding, and signalling, at the receptor. Therefore, while these
findings are exciting, much work remains before the role of the prorenin receptor in
glomerulosclerosis will be understood. An additional point, however, is that recent
in vitro studies indicate that while the clinically available renin inhibitor aliskiren
will likely block angiotensin I formation by receptor-bound renin or prorenin, it
does not appear to be capable of blocking direct signalling at the receptor by these
factors [48].
4.3.4 Intracellular RASs
In 1978, it was reported that tritiated angiotensin II injected into the circulation of
a rat localized to myocardial cell nuclei and mitochondria [49]. Subsequent stud-
ies revealed that hepatic cell nuclei possessed angiotensin II receptors, some asso-
ciated with the nuclear membrane and some with euchromatin and nucleosomal
protein/DNA particles; binding of angiotensin II to these receptors was associated
augmented RNA synthesis. Subsequently, these binding sites were found to be AT-
1-like in character, and binding of angiotensin II to hepatic nuclear sites was associ-
ated with the upregulation of renin and angiotensinogen transcription [50, 51]. Over
the ensuing years, considerable evidence accumulated to support the existence of an
intracellular site of angiotensin action:
(i) Electron-microscopy immunohistology revealed angiotensin II associated
with nuclear euchromatin, consistent with the previously reported chromatin recep-
tors, in unmanipulated animals [52]. (ii) The introduction of angiotensin I or II into
cardiac myocytes produced definite changes in calcium currents; the angiotensin
II effects were blocked by an angiotensin II receptor blocker, the angiotensinogen
effects by an ACE inhibitor. These results also suggest the presence of active renin
in the cells [5356]. (iii) Cells engineered to synthesize intracellular angiotensin II
in the absence of secreted angiotensin (either by introducing an angiotensinogen
construct lacking the signal sequence for secretion or by introducing a construct
encoding octapeptide angiotensin II as an EGF fusion protein) showed marked
proliferation in the absence of extracellular angiotensin II. This effect was not
inhibited by the angiotensin II receptor blocker candesartan but was blocked by
renin antisense, again indicating the existence of an intracellular RAS [5759].

(iv) A second renin transcript encoding a nonsecreted but active renin was identified
by several groups. This renin is found in the adrenal gland and supports aldosterone
synthesis after nephrectomy. It is also upregulated in the rodent left ventricle fol-
lowing myocardial infarction [2125]. (v) It was shown that nonglycosylated renin,
which does in fact circulate to some extent in man, can be internalized by cardiac
myocytes via a heretofore uncharacterized (pro)renin receptor and generate intracel-
lular angiotensin II, thereby producing cardiac pathology [28]. (vi) Angiotensinogen
was shown to be synthesized in some circumstances by glial cells and to reside in
the nucleus. This retention of angiotensinogen by the cells appeared to result from
altered posttranslational phosphorylation [60]. (vii) Immunohistochemical studies
revealed renin, angiotensin [17] and N-terminal ACE immunoreactivity in mesan-
gial cells [61]. (viii) The transfection of cardiac myocytes with a construct encod-
ing a nonsecreted angiotensin II construct led to marked hypertrophy within 96 h;
the same construct injected into mice produced overt myocardial hypertrophy in
the same time period [62, 63]. (ix) Elevated glucose concentrations were demon-
strated to upregulate renin and angiotensinogen synthesis in cultured neonatal car-
diac myocytes, an effect blocked by the renin inhibitor aliskiren [62, 63].
Collectively, these and other results strongly point to the existence of intracellular
RASs in the cardiovascular system. As these results were being developed, a theory
of intracellular peptide action (i.e. intracrine action) was developed to encompass
intracrine factors in the RAS as well as the large and growing number of other
intracrine peptides/proteins. This, in turn, led to the development of an intracrine
physiology and pharmacology, which remain under active investigation [50, 6365].
4.4 Conclusion
Considerable evidence has accumulated over recent decades to indicate that locally
regulated angiotensin II synthesis occurs in many tissues and that this synthesis has
potentially important physiological and therapeutic implications. In addition, phys-
iologically relevant actions of tissue renin, prorenin, angiotensin [17] and other
RAS components have been described, as has the operation of intracellular systems.
Collectively, these findings demonstrate that the renin angiotensin system is more
than a circulating enzymatic cascade regulating blood pressure and volume: It is an
important regulator of tissue biology and structure in the cardiovascular system and
elsewhere.
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Chapter 5
Renin-Angiotensin-Aldosterone System
and Pathobiology of Hypertension
Pierre Paradis and Ernesto L. Schiffrin
Abstract The renin-angiotensin-aldosterone system (RAAS) plays a critical role

in the pathophysiology of elevated blood pressure, both in experimental models and
in humans, in essential hypertension and some forms of secondary hypertension,
such as renovascular hypertension and primary hyperaldosteronism. This is sup-
ported by studies measuring its components, such as renin activity or concentration,
angiotensin II or aldosterone in plasma and tissues, the receptors for these mediators
and their signaling in cells, as well as by inhibition of the different steps in the RAAS
cascade with renin inhibitors, angiotensin Iconverting enzyme (ACE) inhibitors, or
angiotensin receptor blockers (ARBs). The effects of the RAAS on blood pressure
are exerted on blood vessels to induce vasoconstriction, inflammation, growth, and
remodeling and accelerate the progression of both atherosclerosis and arteriosclero-
sis in large vessels and remodeling of resistance arteries, on the kidney to retain salt
and water, a critical effect to induce long-term blood pressure elevation, on the heart
to induce left ventricular hypertrophy and coronary artery disease, and on the brain
to stimulate vasopressin secretion and sympathetic nervous system activity. These
different aspects of the role of the RAAS in hypertension will be reviewed in this
chapter.
Classically, the renin-angiotensin-aldosterone system (RAAS) was described as

a peripheral system regulating blood pressure and water and salt balance
(Chapter 1). Renin secreted from juxtaglomerular (JG) cells in the afferent arterioles
of the kidney cleaves angiotensinogen, an -2-globulin produced and released into
the circulation by the liver, to generate the decapeptide angiotensin (Ang) I. Ang I
is then cleaved by the dipeptidyl carboxypeptidase AngI-converting enzyme (ACE)
in the lungs to generate the vasoactive octapeptide Ang II. Ang II, classically con-
sidered the final mediator of the RAAS, binds to Ang type 1 (AT1 ) receptors. Ang II
increases blood pressure by causing vasoconstriction of blood vessels or, indirectly,
by increasing blood volume through enhanced renal sodium and water reabsorption.
E.L. Schiffrin (B)

Hypertension and Vascular Research Unit, Lady Davis Institute for Medical Research/Sir Mortimer
B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada
e-mail: ernesto.schiffrin@mcgill.ca

36 P. Paradis and E.L. Schiffrin
In addition, by causing release of aldosterone from the adrenal glands which also
causes increased renal reabsorption of salt and water, Ang II contributes to increased
blood pressure. Ang II mediates most of its effects through AT1 receptors, which
are expressed ubiquitously. In rodents there are two AT1 receptor subtypes: AT1a
which is the predominant AT1 receptor subtype in most organs and AT1b which is
highly expressed in the adrenal cortex and the pituitary gland [1]. Studies with AT1a
receptor null (agtr1a) mice have revealed that blood pressure and vascular tone are
regulated by AT1a receptors [2, 3]. Agtr1a mice had a reduced resting blood pres-
sure and no pressor response to Ang II infusion. Ang II also binds to AT2 receptors,
which are highly expressed in fetal tissues, but whose expression decreases dramat-
ically after birth. The density of AT2 receptors is low in adult tissues. AT2 receptors
are expressed in tissues involved in blood pressure regulation, such as the heart,
kidneys, adrenal glands, brain, and vascular smooth muscle (VSMC) and endothe-
lial cells (EC) in the adult [4]. Although there is some evidence that AT2 receptors
may counteract the action of AT1 receptors, their role in blood pressure regulation
remains unclear.
In addition to the peripheral RAAS of renal origin, there are local tissue RAASs
(Chapter 2). More recently, our understanding of the complexity of the RAAS has
been significantly enhanced with the finding that pro-renin is more than a precursor
protein but also an active molecule, and the discovery of renin receptors (Chapter
3), ACE2 (Chapter 11), and new vasoactive peptides such as Ang IV, Ang 1-7, and
Ang 1-12 (Chapter 11), and new receptors like Ang 4R/IRAP and Ang 1-7R/Mas,
as well as the rediscovery that Ang III may play important roles in the brain and in
the kidney that had not been detected in the past.
From the initial finding of the different components of the RAAS, it has been
assumed that this system is involved in the development and maintenance of hyper-
tension. This was first recognized in acquired disorders, particularly in renovascu-
lar hypertension for renin and Ang II, and primary aldosteronism in the case of
aldosterone, and also in essential hypertension (reviewed in reference 2). Both Ang
II, the main peptide effector of RAAS, and aldosterone exert effects that partici-
pate in the mechanisms leading to the development of hypertension by acting on
the vasculature, the kidneys, and the central and peripheral nervous system. Ang II
and aldosterone also act on the heart beyond their effects on the coronary vessels.
These effects on the myocardium are part of the target organ damage associated with
hypertension.
5.1 Blood Vessels
Essential hypertension is characterized by increased peripheral vascular resistance

to blood flow [5], occurring mostly through energy dissipation in small resistance
arteries with a reduced lumen diameter. It is important to note that according to the
law of Poiseuille, flow resistance is inversely related to the fourth power of the ves-
sel radius, and therefore, small decreases in the diameter of the lumen significantly
increase resistance. Small resistance arteries with a lumen diameter of 100300 m
play an important role in the development of hypertension [6], and contribute to
5 Renin-Angiotensin-Aldosterone System and Pathobiology of Hypertension 37
its complications [7], to myocardial ischemia [810], stroke [11], and renal failure
[12]. In essential hypertension, resistance arteries undergo eutrophic remodeling
characterized by reduced outer diameter and lumen and increased media-to-lumen
ratio, but with unaltered media cross-sectional area [1320]. Eutrophic remodeling
has been most often observed in animal models of hypertension with an activated
RAAS [4, 21, 22]. This remodeling is characterized in humans by the absence of
VSMC hypertrophy or hyperplasia [23] but with rearrangement of VSMCs around
the smaller lumen [2426]. The VSMC rearrangement may result from increased
constriction [27, 28] due to activation of the RAAS and/or the sympathetic nervous
system or secretion of growth factors such as endothelin-1 (ET-1), which becomes
embedded in an increased extracellular matrix, also resulting from the action of
RAAS components, such as Ang II or aldosterone, or other agents. Hypertrophic
remodeling, which is characterized by increased media-to-lumen ratio and media
cross-sectional area, has been observed in small arteries of patients with renovas-
cular hypertension [29], hypertensive diabetic subjects [30, 31], and acromegalic
patients [32]. This remodeling is associated with VSMC hypertrophy without evi-
dence of hyperplasia. It is noteworthy that VSMC hyperplasia and hypertrophy have
been shown to contribute to vascular remodeling in animal models of hyperten-
sion, such as spontaneous hypertensive rats (SHRs), stroke-prone SHR (SHR-SP),
and Ang II-induced hypertension in rodents [3337]. We demonstrated that Ang II
induced proliferation (hyperplasia) and growth (hypertrophy) of cultured vascular
VSMCs isolated from resistance arteries from subcutaneous gluteal biopsies from
human healthy subjects [38] through AT1 receptors via the ERK-dependent sig-
naling pathway and increased generation of reactive oxygen species (ROS) [39].
Furthermore, Ang II induced growth and proliferation of VSMCs through a cross-
talk between AT1 receptors and epidermal growth factor (EGF), platelet-derived
growth factor (PDGF), and insulin-like growth factor (IGF) receptors [28]. Aortic
media thickening in animal models of hypertension is accompanied by re-expression
of fetal genes in VSMCs associated with a shift of VSMCs from a contractile to
a synthetic phenotype and/or expansion of preexisting immature VSMC popula-
tion [4042], and accordingly, in TGRen2 transgenic rats overexpressing the mouse
Ren2 gene, Ang II induced re-expression of fetal muscle genes (SM-MyHC and
MyHC-2) and EIIIA-fibronectin (FN) in aortic VSMCs, which may play a role in
the changes observed in VSMC phenotype [40] and vascular stiffness (see below).
Apoptosis in the periphery of small arteries triggered by Ang II, combined with
growth of VSMCs toward the lumen, may contribute to the VSMC rearrangement in
eutrophic remodeling [28, 43]. However, Rizzoni et al. observed apoptosis in small
mesenteric arteries of older SHRs while no apoptosis was present in younger rats in
which eutrophic remodeling was already present [44]. Interestingly, whereas Ang
II infusion induced apoptosis in aortic VSMCs of Wistar rats, AT1 or AT2 receptor
blockers did not prevent but actually enhanced apoptosis [45], suggesting a role for
both receptors in this process. Although the role of AT2 receptors is still unclear,
they may counteract and fine-tune AT1 pro-proliferative actions [46].
Fibrosis of the media is observed in hypertension in blood vessels in both
eutrophic and hypertrophic remodeling [31], and may contribute to increased resis-
tance by augmenting vasculature stiffness [47]. Vascular fibrosis is characterized by

the accumulation of extracellular matrix components such as collagen, FN, fibrillin,
and proteoglycans in the media and perivascularly [48]. Ang II regulates the syn-
thesis and degradation of collagen in the vascular media. Results from in vivo stud-
ies have indicated that Ang II through AT1 receptors induced collagen deposition
in the media [40, 49]. In vitro studies further demonstrated that Ang II increased
expression of collagen in VSMCs and fibroblasts through AT1 receptors [50, 51].
In addition, Ang II decreased the expression of enzymes that degrade collagen,
the matrix metalloproteinases (MMPs), and stimulated production of the tissue
inhibitors of MMPs (TIMPs) [52, 53]. Ang IIAT1 receptor-dependent signaling
pathways upregulated the expression of a fetal extracellular matrix gene, EIIIA-FN
[48]. Increased levels of Ang II in TGRen2 transgenic rats increased EIIIA-FN in
aortic SMCs, thus contributing further to vascular stiffness [40]. The increase in FN
preceded the rise in blood pressure in Ang II-dependent hypertensive models [48].
The profibrotic effects of Ang II via AT1 receptors were mediated by transforming
growth factor (TGF) [54] or connective tissue growth factor (CTGF) [55, 56].
Increased vasoconstriction may contribute to development of hypertension.
Indeed, we have observed exaggerated vasoconstriction response to Ang II in
resistance arteries of patients with essential hypertension [16]. Increased contrac-
tile sensitivity has also been found in rodent models of hypertension [57]. This
enhanced contractile response may be mediated by aldosterone-induced increase
in AT1 receptor expression [58, 59] and/or through enhanced signaling responses
by the RhoA/Rho kinase-dependent pathways in VSMCs [57].
Endothelial dysfunction may also play a role in the increases in peripheral resis-
tance found in both experimental and human hypertension. We and others have
observed endothelial dysfunction in humans [60, 61] and in SHRs [60, 62]. This
altered endothelial function is mainly dependent on reduced bioavailability of nitric
oxide (NO), as reported in human subjects with essential hypertension [60, 63, 64]
and in animal models such as SHR-SP and Ang II-induced hypertension in rabbits
[65, 66]. Decrease in NO bioavailability could result from a reduction in synthesis
or an increase in degradation of NO. Inhibition of NO synthesis could result from
increases in asymmetric dimethylarginine (ADMA), which is a potent endogenous
inhibitor of NO synthase (NOS) [67]. ADMA is produced by methylation of argi-
nine by protein arginine N-methylytansferase (PRMT) and release by proteolysis.
Normally, most of ADMA is degraded by dimethylamine dimethylaminohydrolase
(DDAH) and the remaining is excreted in the urine. Increased levels of ADMA have
been shown in hypertensive subjects [64, 68, 69] and in SHR [63]. Interestingly, the
levels of ADMA and NO were, respectively, decreased and increased by blockade
of the RAAS independently of blood pressure [63, 69]. Ang II-induced oxidative
stress may contribute to decrease in biovailability of NO [70, 71]. Ang II induces
the production of superoxide ( O2 ) mainly through activation of reduced nicoti-
namide adenine dinucleotide phosphate (NADPH) oxidase, which is expressed in
all vascular layers including ECs, VSMCs, and adventitial fibroblasts [70], although
other enzymes such as xanthine oxidase, the respiratory chain, etc., probably also
contribute to different degrees. Increase in ROS was documented in humans with

essential, renovascular, and malignant hypertension and in women with pre-
eclampsia [63, 7274]. In experimental models of hypertension, increases in oxida-
tive stress in different tissues including the vasculature have been demonstrated in
deoxycorticosterone acetate (DOCA)-salt mice, SHRs, and SHR-SPs [7578]. We
observed that basal and Ang II-induced NADPH oxidase-driven O2 production
increased in VSMCs from SHRs with the level of blood pressure elevation [75]. Fur-
thermore, we and others found that blockade of the RAAS, for example, with the
AT1 receptor blocker (ARB) valsartan, decreased the production of ROS in SHR-
SP and in Dahl salt-sensitive hypertensive rats [7779]. Decreases in NO bioavail-
ability could also be caused by oxidation of NO to peroxynitrate ( ONOO ) by
O . In addition, Ang II-induced generation of O and ONOO may oxidize
2 2
tetrahydrobiopterin (BH4 ), an essential co-factor of endothelial NOS (eNOS), to
7,8-dihydrobiopterin (BH2 ). This will cause uncoupling of eNOS and change its
state from one in which it produces NO to one in which it produces O2 . BH4
levels and the rate-limiting enzyme for the de novo synthesis of BH4 , guanyltriphos-
phate cyclohydrolase (GTPCH) I, as well as endothelium-dependent acetylcholine-
induced vascular relaxation, were decreased in DOCA-salt hypertensive mice [76].
Interestingly, endothelium-specific overexpression of GTPCH I restored endothelial
function in DOCA-salt mice. Several other studies have demonstrated that treatment
with BH4 decreased blood pressure and improved endothelial function in Dahl salt-
sensitive hypertensive rats and in Ang II-induced hypertension in rats [7981].
Peripheral resistance is modulated by up to slightly under 20% by the den-
sity of capillaries. Factors affecting angiogenesis such as hypoxia and inflam-
mation modify capillary density and therefore blood pressure. Most forms of
human and experimental hypertension are associated with decreased density of
microvessels (rarefaction), which can increase blood pressure further and exacer-
bate hypertension-induced end-organ damage [28, 82]. Apoptosis of ECs may also
be involved in microvascular rarefaction, contributing thus to hypertension [83, 84].
This could be mediated in part by Ang II, since inhibition of the RAAS with an ACE
inhibitor has been shown to increase microvascular density [82].
Vascular remodeling and endothelial dysfunction are accompanied by local
inflammation, which may contribute to the development of hypertension and its
complications [28]. Inflammation is actually a normal process involved in recov-
ery of tissue integrity, but if repair is not well regulated, this may lead to persistent
changes and tissue damage. ROS have been implicated in all the stages of inflam-
mation. Growing evidence indicates that low-grade inflammation plays a significant
role in the pathophysiology of RAAS-induced high blood pressure and its complica-
tions. Ang II-induced O2 by activation of NADPH oxidase in the vasculature may
be an early step in the initiation of local inflammation. Furthermore, Ang II has been
implicated in all the subsequent steps of inflammation characterized by an increase
in vascular permeability, leukocyte recruitment, and activation of tissue repair [28].
Ang II increased vascular permeability by inducing the synthesis of prostaglandins
(PGs) and vascular endothelial growth factor (VEGF) in VSMCs and ECs through
AT1 receptors [85]. Ang II-induced leukocyte recruitment and activation via AT1
receptors, by inducing the expression of selectins (P and E) [86, 87], integrins (2
and 4) [86], intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion
molecule (VCAM)-1 [87], cytokines such as monocyte chemotactic protein (MCP)-
1, interleukin (IL)-6, IL-8, IL-18, osteopontin (OPN), tumor necrosis factor (TNF)-
[8892], and chemokines such as cytokine-inducible neutrophil chemoattractant
(CINC), keratinocyte-derived chemokine (KC), macrophage inflammatory protein
(MIP)-2, and CC chemokine ligand 5 (CCL5) [89, 90, 93]. Furthermore, Ang
II induces low-grade inflammatory effects independently of its blood pressure-
raising actions. Inhibition of VEGF by soluble VEGF receptor 1 (sFlt-1) gene
transfer attenuated Ang II infusion-induced inflammation and vascular remodel-
ing in mice without normalization of blood pressure [94]. A sub-pressor dose of
Ang II induced leukocyte adhesion in mesenteric arteries via pressor-independent
mechanisms [89].
Ang II may also induce inflammation by acting directly on lymphocytes. Nataraj
et al. observed that T and B cells and macrophages isolated from the spleen express
AT1a receptors and that in vitro Ang II regulated the proliferation of wildtype but
not agt1ar/ splenic lymphocytes [95]. More recently, it was demonstrated that
T lymphocytes are required for Ang II to induce vascular remodeling [96]. Ang
II-induced vascular remodeling was impaired in rag1/ mice, which are deficient
in both T and B lymphocytes, although expression levels of aortic AT1 and AT2
receptors were unaltered. The vascular effects of Ang II were restored by trans-
fer of T but not B cells. Finally, the last step of inflammation, tissue repair, which
is a normal process that restores tissue integrity, may be impaired by the action
of Ang II. Indeed, Ang II causes VSMC growth, proliferation and apoptosis, and
vascular fibrosis, leading to vascular remodeling and hypertension (see above). Fur-
thermore, Ang II-induced inflammation is a major contributor to the progression of
atherosclerosis. The ACE inhibitor (ACEi), quinapril, blunted the increased expres-
sion of the CC chemokine MCP-1 and macrophage infiltration in the neointima of
injured femoral artery in a rabbit model of accelerated atherosclerosis [97]. The
ARB ibesartan reduced both the progression of the lesions and the expression of
CC chemokines such as MCP-1 and macrophage inflammatory protein (MIP)-1
and CXC chemokines (MIP-2 and KC) in artherosclerotic lesions of apoE/ mice
[98]. The ARB losartan decreased both intima proliferation and the expression of
P-selectin and macrophage infiltration in aorta of hypercholesterolemic rabbits [99].
The ARB irbesartan reduced the serum level of O2 ,VCAM-1, and TNF- in nor-
motensive patients with coronary artery disease (CAD) who had undergone coro-
nary artery bypass graft, percutaneous transluminal coronary angioplasty, or both
prior to the study. The ARB candesartan reduced the serum levels of two proin-
flammatory molecules in hypertensive patients, the acute-phase reactant C-reactive
protein (CRP) produced by the liver and adipocytes and CD40 ligand expressed by
activated T lymphocytes, independently of blood pressure reduction [100]. In addi-
tion, Ang II-induced inflammation may further exacerbate artherosclerotic coronary
artery disease (CAD) or stroke by causing atherothrombosis, which is characterized
by an unpredictable, sudden rupture or erosion/fissure of an atherosclerotic plaque,
which leads to platelet activation and thrombus formation. Plasminogen activator

inhibitor (PAI)-1, the major inhibitor of fibrinolysis, is increased in atherosclerotic
plaques [101, 102], and Ang II has been demonstrated in vitro to induce expres-
sion of PAI-1 in endothelial cells [103, 104] and in vivo to cause a rapid increase
in circulating PAI-1 in normotensive and hypertensive patients [105]. Inhibition of
the RAAS with the ACE inhibitors ramipril or fosinopril decreased, respectively,
the plasma levels of PAI-1 in patients with acute CAD [106] and in type 2 dia-
betic hypertensive patients [107]. Administration of the ARB valsartan to diabetic
hypertensive subjects corrected structural remodeling of small arteries [108] and
also reduced proinflammatory cytokines [109]. Interestingly, in the diabetic hyper-
tensive subjects, the ARB treatment was associated with upregulation of AT2 recep-
tors, which could contribute to the beneficial effects of valsartan on remodeling and
inflammation [110].
Renin receptors have been described in different tissues including blood ves-
sels [111], but their significance with respect to blood pressure elevation remains
unclear. This subject is treated extensively in Chapter 3.
So far, this chapter has concentrated on effects attributable to actions of Ang II.
However, aldosterone, whose secretion by the adrenal glomerulosa is stimulated by
Ang II, has important vascular effects (for more complete review see reference 112).
These affect not only the endothelial layer [113] and the media but also the adventi-
tia. Aldosterone binds to the mineralocorticoid receptor (MR) and has proliferative,
proinflammatory, and profibrotic actions that are mediated in part via its genomic
effects and in part via its nongenomic actions. These effects in part appear to medi-
ate vascular actions usually attributed to Ang II [114116]. Aldosterone appears to
contribute to vascular actions of angiotensin II in part via upregulation of Ang recep-
tors [58, 117] and other components of the RAS, not only in the vasculature but also
in the brain [118]. It may also act in concert with Ang II to contribute to enhance
its actions, area which is under active research. Aldosterone exerts its nongenomic
effects by stimulating oxidative stress mainly through activation of NADPH oxidase
and stimulation of MAPK (ERK 1/2) and other kinases [119]. Through genomic
effects, aldosterone and other mineralocorticoids stimulate the expression of differ-
ent proteins and peptides, including ET-1, which contributes to the oxidative stress,
proliferation, inflammation, and fibrosis [120]. Increased oxidative stress partici-
pates in the endothelial dysfunction associated with effects of aldosterone [121],
as NO is scavenged by ROS. The contribution of aldosterone to the hypertensive
process is highlighted by the fact that it participates not only when aldosterone is
produced in excess by an adrenal adenoma, which will not be discussed here [122],
but also in essential hypertension, where there may be inappropriate secretion of
aldosterone, particularly in resistant hypertension [123] and among obese hyperten-
sive subjects [124]. Treatment of hypertensive subjects with blockers of MR, such as
eplerenone, resulted in lowering of blood pressure (demonstrating the participation
of aldosterone in blood pressure elevation) but as well decreased stiffness of large
and small arteries of hypertensive patients [125]. Typically, collagen and fibronectin
deposition in the media was reduced by eplerenone, whereas elastin was enhanced
and vessels became more distensible.
5.2 The Kidney

The effects of Ang II and aldosterone that impact on the kidney to contribute to the
pathophysiology of hypertension will be dealt with only briefly. True renovascu-
lar hypertension in humans, in which increased renin secretion occurs in response
to renal artery stenosis, or renin-secreting tumors, or those forms of experimen-
tal hypertension in which renin secretion by the juxtaglomerular cells is increased,
such as Goldblatt hypertension in rodents or dogs, will not be addressed. Rather,
it is the effects that the RAAS has on the kidney, which may lead to hypertension,
and more specifically that of Ang II, which will be described. The specific effects
of aldosterone on salt and water balance are dealt with in Chapter 15.
The role of the kidney in long-term control of blood pressure and in the develop-
ment and maintenance of hypertension was underlined by the computer modeling
carried out by Arthur Guyton [126], and its relation to Ang II clarified in large mea-
sure by studies of John Hall [127] and others. Infusion of Ang II impairs pressure
natriuresis, and blood pressure becomes highly sensitive to sodium intake, resulting
in blood pressure elevation in the presence of small increments in sodium in the
diet, and this is corrected by inhibition of Ang II generation by an ACE inhibitor
(Fig. 5.1) [128]. This is particularly important in view of the fact that the kidney
has large amounts of Ang II generated locally [129], which may be increased in
hypertension as suggested by studies with RAAS blockade selectively administered
Fig. 5.1 Chronic relationships between blood pressure and sodium intake and excretion in dogs
with a normal RAS, after blockade of Ang II formation with an ACE inhibitor and after continuous
infusion of a low dose of Ang II (5 ng/kg per minute) to prevent suppression of circulating Ang
II upon increased sodium intake. Inability to modulate Ang II levels decreases the slope of pres-
sure/natriuresis relationship, causing marked salt sensitivity of blood pressure. Reproduced from
[127] with permission
to the kidney, which improve natriuresis [127]. Ang II, through its hemodynamic
and tubular effects on the kidney (for review see reference 130), and aldosterone,
through its sodium-retaining action, will contribute thus to blood pressure eleva-
tion. The similarity of what happens with Ang II infusion, which can be reversed by
ACE inhibitors as shown in Fig. 5.1 [128], and the displacement to the right of the
blood pressure/urinary volume output relationships in both salt-sensitive and non-
salt-sensitive essential hypertension compared to normal as shown in Fig. 5.2 [131]
suggests that indeed Ang II plays this role in renal hemodynamics and natriuresis,
leading to blood pressure elevation. Interestingly, studies by T. Coffman and his
group have demonstrated the critical importance of the AT1a receptor in the kidney
in blood pressure elevation in response to Ang II infusion in mice using gene dele-
tion and cross-transplantation studies (Fig. 5.3) [132]. From these studies, it would
appear that in mice, kidney AT1a receptors that seem to be tubular rather than vas-
cular play a more important role in blood pressure elevation induced by Ang II than
systemic AT1a receptors. Whether this also applies to other species remains to be
determined.
Fig. 5.2 Blood pressure/volume output relationships in normal conditions to the left or in salt-
sensitive and non-salt-sensitive essential hypertension to the right. Reproduced from [131] with
permission
A word should be said about renin receptors and the kidney [111]. These have
been identified in the kidney, but little is known about their importance in relation to
blood pressure control by the RAAS. However, it has been suggested that renin may
Fig. 5.3 (a) Kidney cross-transplantation groups used in the study by Coffmans group. Wild-type
(+/+) or AT1a receptor-deficient (/) mice were transplanted with kidneys from either AT1a (+/+)
or AT1a (/) mice. Systemic knockout (KO) mice express AT1a receptors only in the kidney since
they have received a kidney transplant from AT1a (+/+) mice. Kidney KO animals express AT1a
receptors in all tissues except the kidney, since they have been transplanted with kidneys from
AT1a (/) mice. Total KO animals lack AT1a receptors completely. (b) Daily blood pressures in
cross-transplanted mice during 21-day Ang II infusion. By day 12 of Ang II infusion, the severity
of blood pressure elevation in systemic KO reaches that of the wild-type mice. Absence of renal
AT1a receptors in kidney KO mice blunts the development of Ang II-induced blood pressure ele-
vation. Total KO blood pressure shows minimal response to Ang II infusion ( P0.03 vs wild
type; P<0.008 vs systemic KO; P<0.006 to 0.0001 vs wild type). Reproduced from [130] with
permission
act directly via these receptors to induce fibrosis in the kidney [133], which could
contribute to nephroangiosclerosis and blood pressure elevation. Although some
evidence was produced regarding a potential role in diabetic nephropathy [134],
recent data suggest that in contrast, in Goldblatt hypertension in rats, renin recep-
tors do not seem to play a role, or at least the so-called handle-region peptide renin
receptor inhibitor has no effect on hypertensive nephrosclerosis [135].
5.3 The Heart

Left ventricular hypertrophy (LVH) is one of the prototypical manifestations of tar-
get organ damage found in hypertension and is associated with increased risk of
cardiovascular events [136]. Whether LVH is only a consequence of elevated blood
pressure or also results in part from the effects of Ang II has been a matter of contro-
versy for some time. In vitro, Ang II causes cardiac myocyte hypertrophy [137], and
treatment of hypertension with blockers of the RAAS results in regression of LVH
[138, 139]. High-level (200- to 400-fold) overexpression of AT1 receptors in the
heart under control of the alpha-myosin heavy chain (MHC) promoter resulted
in ventricular hypertrophy only in mice older than 1.5 months and rapidly pro-
gressed to heart failure associated with myocyte apoptosis and fibrosis and death
[140]. Accordingly, it has been concluded that Ang II may indeed participate in the
induction of LVH through its action on cardiomyocytes and fibroblasts, stimulat-
ing the hypertrophy of the former and fibrosis by its action on the latter. However,
some studies have provided quite definitive evidence that this may not be the case
at least in mice. Coffmans group in their studies of gene deletion of AT1a receptors
and cross-transplantation showed that mice with renal AT1a receptors and no sys-
temic (therefore cardiac) AT1a receptors, when infused with Ang II, develop high
blood pressure and LVH [141]. In contrast, mice with no renal but systemic and
cardiac AT1a receptors infused Ang II develop neither hypertension nor LVH. This
suggests that Ang II acting on AT1a receptors in the heart does not induce LVH,
whereas elevation of blood pressure even in absence of AT1a receptors in the heart
will induce LVH. Production of Ang II in the heart using the MHC promoter to tar-
get the cardiac expression of an engineered fusion protein that directly releases Ang
II produced transgenic mice with 20- to 50-fold elevation of cardiac levels of Ang
II with no detectable increase in circulating Ang II [142]. These mice developed
slight interstitial fibrosis but no cardiac hypertrophy at 3 months of age. An engi-
neered fusion protein was also used that released a form of Ang II that could not be
degraded, which resulted in extremely high levels of cardiac Ang II and in spillover
into the circulation. Cardiac hypertrophy did not occur despite the very high levels
of cardiac Ang II until blood pressure rose in response to the increases in circulating
Ang II. Finally, some recent studies have demonstrated that signaling pathways for
pressure-mediated and direct (Ang II-mediated?) effects through AT1 receptors may
be different. Mice in which the gene for Gq /11 has been deleted specifically in car-
diomyocytes did not develop myocardial hypertrophy after pressure overload [143],
whereas mice in which an AT1 receptor devoid of the Gq -coupling intracellular
loop (i2m) has been overexpressed in the heart had more ventricular hypertrophy
than those in which the nonmutated receptor was overexpressed [144]. Thus, most
of the evidence does not support a direct role of the RAAS in LVH. LVH can, how-
ever, be improved by RAAS inhibition even if the RAAS activation may not be the
primary cause of cardiac hypertrophy [145]. However, direct activation of cardiac
AT1 receptors may contribute to the development of LVH through alteration of car-
diac repolarization, which is a major risk factor for ventricular arrhythmias and sud-
den death. Detailed analysis of ventricular repolarization parameters in mice with
specific cardiomyocyte AT1 receptor overexpression revealed an increased

incidence of cardiac arrhythmia associated with delayed repolarization in 50-day-
old mice, before the development of cardiac hypertrophy [146]. Increased incidence
of cardiac arrhythmias has also been observed in mice with cardiac-restricted ACE
and in AT1 i2m mutant receptor transgenic mice [144, 147].
Aldosterone as well as Ang II will have effects on the heart, which contribute
to the pathophysiology of hypertension, namely via its effects on cardiac fibrosis
and inflammation [50, 148]. The effects of aldosterone on the heart have been par-
ticularly underlined by the clinical studies with mineralocorticoid receptor blockers
such as spironolactone in heart failure in the RALES trial [149] and eplerenone
postmyocardial infarction in the EPHESUS trial [150]. Interestingly, it appears that
effects of aldosterone on the heart require a functional AT1 receptor [151, 152]. It
has been suggested that gene inactivation of the mineralocorticoid receptor in car-
diomyocytes was associated with cardiac fibrosis and heart failure in mice, which
could be reversible if treated with spironolactone, although this work remains con-
troversial and unexplained [153].
With respect to the coronary circulation, what was already reviewed in the sec-
tion on the blood vessels is applicable as well to coronary vessels. Randomized
clinical trials have shown that RAAS blockade with ACE inhibitors or ARBs pro-
tects patients with and without hypertension from cardiovascular events [154, 155].
The effects of Ang II on progression of atherosclerosis, on PAI-1, on thrombosis and
inflammation, on MMPs and plaque rupture that have already been referred may be
underlying the benefits derived from RAAS inhibition at the level of the coronary
circulation, which will not be reviewed here in detail (for review see reference 156).
5.4 The Brain and the Sympathetic Nervous System
The brain and the sympathetic nervous system have been implicated in the patho-
physiology of hypertension by well-founded evidence for many years [157]. Brain
renin, as well as all other components of the RAS, including ACE, AT1 and AT2
receptors, angiotensinogen, Ang II, Ang III, and Ang IV have been demonstrated in
the brain [158]. Ang II induces pressor and dipsogenic responses and vasopressin
secretion after injection into the brain [159, 160]. Moreover, there are Ang II recep-
tors in the circumventricular organs (which are devoid of a bloodbrain barrier and
therefore accessible to circulating Ang II and include the subfornical organ or SFO,
organum vasculosum lamina terminalis or OVLT, area postrema on the floor of the
fourth ventricle), and are in the vicinity and connected to cardiovascular control
nuclei of the brain [161], as well as in other regions of the brain including the cor-
tex and the cerebellum and the choroid plexus. These findings have provided the
anatomic and physiological basis involvement of the RAAS in the role that the cen-
tral nervous system (CNS) plays in blood pressure control and elevation [162]. In
part, these central effects of Ang II are mediated via AT1 receptors and increased
activity of the sympathetic nervous system (SNS) [163]. AT2 receptors may coun-
teract some of the actions mediated by AT1 receptors [164].
In addition to the brain, Ang II induces increased release of norepinephrine (NE)

from nerve endings of the SNS in the periphery, and epinephrine from the adrenal
medulla, and as well affects baroreflex function, an important contributor to blood
pressure regulation [165]. These effects may play a role in blood pressure eleva-
tion in both experimental animals and humans. Increased oxidative stress occurs in
the circumventricular organs in the brain in response to Ang II administered both
centrally and peripherally [166, 167]. Blockade of generation of ROS in these cir-
cumventricular organs inhibits both central and peripheral Ang II-induced blood
pressure elevation [168, 169].
Mineralocorticoid receptors are also present in the hypothalamus and elsewhere
in the brain, and aldosterone induces effects on the hypothalamus, which have been
implicated in heart failure through interactions with other components of the RAS
[170]. Indeed, aldosterone exerts effects on the brain that worsen manifestations
of heart failure in rodents in part through upregulation of AT1 receptors [118], as
already demonstrated many years ago in peripheral blood vessels [58].
The complex mechanisms whereby the CNS, the SNS, and the adrenal medulla
may play a role in hypertension in response to the effects of Ang II and aldos-
terone are beyond the scope of this short review. It should be noted, however, that
Ang II-like immune reactivity has been found in many areas of the brain and brain
stem such as the anterior and middle hypothalamus, basal ganglia, locus coeruleus,
nuleus tractus solitarius, and reticular formation of SHR [171], and that increased
AT1 receptors have been found in circumventricular organs and cultured brain neu-
rons of SHR [172, 173]. Moreover, intracerebroventricular administration of cap-
topril, an ACE inhibitor, or saralasin, an Ang receptor blocker, or an antisense to
angiotensinogen or to AT1 receptors in intact hypertensive rats, or peripherally in
anephric rats, results in blood pressure lowering [174178]. Thus, in experimental
hypertensive models, Ang II stimulation of brain centers appears to play an impor-
tant role. To what degree this occurs in humans has not been established.
5.5 Conclusion
The RAAS is involved in the pathophysiology of hypertension at the level of ves-
sels, the heart, the kidney, and the central and peripheral nervous system and par-
ticipates in the complications of hypertension. Both Ang II and aldosterone have
been proven to play roles in these different tissues and organ systems. Thus, it is not
surprising that treatment of patients with hypertension with blockers of the RAAS,
be it renin inhibitors, ACE inhibitors, ARBs, or mineralocorticoid receptor block-
ers, results in improved blood pressure control and better outcomes in humans with
essential hypertension, beyond the secondary forms of hypertension such as renin-
secreting tumors, true renovascular hypertension (with increased renin secretion due
to a renal artery stenosis), or primary aldosteronism. However, there are many com-
ponents of the RAAS whose exact contribution to pathophysiology remains unclear,
including renin receptors, ACE2, Ang 1-12, Ang 1-7, Ang III, and Ang IV. The
participation of these, and the development of treatments thereof, should help not
only our understanding but also the benefits that patients with hypertension may
derive from interference with the RAAS.
Acknowledgments The work of the authors was supported by grants 37917 and 82790 from the
Canadian Institutes of Health Research (CIHR) and by the Canada Fund for Innovation and the
Canada Research Chairs program of the Government of Canada, all to ELS.
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Chapter 6
AT1 Receptors, Angiotensin Receptor Blockade,
and Clinical Hypertensive Disease
Robert M. Carey
Abstract The renin angiotensin system (RAS) is a coordinated hormonal cascade,

the major effector peptide of which is angiotensin II (Ang II). Ang II binds to
one of two principle receptors, AT1 (AT1 R) and AT2 (AT2 R). AT1 Rs mediate the
vast majority of biological actions of the RAS, almost all of which are potentially
detrimental. This chapter focuses on new developments in our knowledge of AT1 Rs
and the actions of angiotensin receptor blockers (ARBs) in the treatment of hyper-
tension. Several novel mechanisms have been described whereby AT1 Rs mediate
detrimental actions in cardiovascular and renal function. These include [1] Ang II-
independent activation of AT1 Rs, which respond only to inverse agonist administra-
tion [2]; AT1 R-activating autoantibodies, potentially important in the pathogenesis
of pre-eclampsia and other conditions associated with inflammation [3]; G protein
receptor-interacting proteins which may modulate the functional activity of AT1 Rs
[4]; and receptor cross-talk/receptor dimerization. New considerations of ARBs for
the treatment of hypertension include [1] renal tissue RAS function independently
of the systemic circulation [2], AT2 R activation in response to AT1 R blockade [3],
pleiotropic actions of ARBs, and new data on the use of combination RAS blockade.
The results of several major new clinical trials are discussed and placed in context
with existing antihypertensive therapy.
6.1 Introduction
The renin angiotensin system (RAS) is a coordinated hormonal cascade of major
critical importance in the regulation of blood pressure. The principal effect or pep-
tide of the RAS is angiotensin II (Ang II), which acts by binding to one of two major
angiotensin receptors, type-1 (AT1 R) and type-2 (AT2 R) (Fig. 6.1) [1]. AT1 Rs medi-
ate the vast majority of the biological actions of Ang II, including vasoconstriction,
R.M. Carey (B)

Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia
Health System, Charlottesville, VA, USA
e-mail: rmc4c@virginia.edu

60 R.M. Carey
Agt
Renin
Inactive
Ang I fragments
ACE
Inhibits Ang II Bradykinin
Substance P
AT1R AT2R
Fig. 6.1 Schematic diagram of the renin angiotensin system. Agt angiotensinogen; Ang I
angiotensin I; Ang II angiotensin II; ACE angiotensin-converting enzyme; AT1 R angiotensin
type-1 receptor; AT2 R angiotensin type-2 receptor
antinatriuresis, sympathetic nervous system activation, aldosterone, vasopressin and

endothelin secretion, and vascular smooth muscle hypertrophy, migration, prolifera-
tion, and growth, all of which act in concert to raise blood pressure (BP). In addition
to a rise in BP, AT1 R activation induces a number of detrimental effects in cardio-
vascular and renal tissues (Fig. 6.2), including cytokine production by monocytes
and macrophages, leading to inflammation; plasminogen activator inhibitor-1 (PAI-
1) biosynthesis, platelet activation, aggregation and adhesion, leading to thrombosis;
collagen biosynthesis leading to fibrosis; and low-density lipoprotein transport lead-
ing to atherosclerosis. These tissue actions of Ang II via AT1 Rs have been attributed
in large part to a common mechanism: the production of reactive oxygen species,
especially superoxide anion via stimulation of NAD(P)H oxidase with accompa-
nying nitric oxide destruction [2]. Other actions of Ang II through AT1 Rs include
increased cardiac contractility, cardiac and vascular remodeling, and reduction in
vascular compliance. These detrimental actions of Ang II can be offset to some
extent by AT1 R-mediated short-loop negative feedback suppression of renin biosyn-
thesis and secretion at renal juxtaglomerular cells.
In contrast to the pressor and tissue destructive mechanisms of Ang II via AT1 Rs,
Ang II activation induces opposite effects via AT2 Rs, including vasodilation of both
resistance and capacitance vessels, natriuresis and inhibition of cellular proliferation
and growth [3, 4]. However, the relative balance between AT1 R and AT2 R functions
may be influenced by receptor expression patterns in tissues. Whereas AT1 Rs are
highly expressed in the cardiovascular, renal, endocrine, and nervous systems in
adults, AT2 R expression is quantitatively less and its tissue distribution more limited
than that of AT1 Rs. The extent to which AT2 Rs are functionally counterregulatory
to AT1 Rs and play a role in the pathophysiology of hypertension continues to be a
subject of intensive study.
6 AT1 Receptors, Angiotensin Receptor Blockade, and Clinical Hypertensive Disease 61
Renal Na+ Cytokines/

Abnormal retention inflammation
vasoconstriction
PAI-1/
Activate SNS thrombosis
Aldosterone
Platelet
Angiotensin II aggregation/
Vasopressin adhesion
Superoxide
Endothelin production/
NO destruction
Vascular Collagen/
Cardiomyocyte smooth muscle
growth fibrosis
growth
Cardiac and vascular

remodeling
Fig. 6.2 Deleterious actions of angiotensin II. SNS sympathetic nervous system; NO nitric
oxide; PAI-1 plasminogen activator inhibitor 1
Hypertension is recognized as one of the leading risk factors for human morbid-
ity and mortality and, on a worldwide basis, is ranked third as a cause of disability-
adjusted life years (DALYS) [5]. The enormity of the problem of hypertension is
underscored by the fact that one-quarter of the worlds adult population, totaling
nearly one billion individuals, had hypertension in the year 2000, and this number
is predicted to increase to 29% by the year 2025 [6]. Despite the huge burden of
disease and the availability of several different classes of antihypertensive pharma-
cological agents, relatively few patients achieve their target BP level. In the United
States during 20032004, only 33% of hypertensive patients had controlled BP and
only 64% of patients treated for hypertension achieved BP control [7]. Even more
strikingly, in five European countries approximately 70% of hypertensive patients
do not meet their BP targets [8]. As a major risk factor for myocardial infarction,
congestive heart failure, stroke, and end-stage renal disease, all of which convey
risk of significant morbidity and mortality, hypertension is an enormous public
problem [9].
The difficulty in controlling hypertension is related, at least in part, to the com-
plex pathogenesis of hypertension and related cardiovascular disease. Multiple sig-
naling pathways and redundant feedback mechanisms, both positive and negative,
contribute to the hypertensive disease process, which is even more confounded by
the interrelationship of hypertension with associated diseases such as diabetes and
renal dysfunction. The RAS plays an important role not only in the control of BP but
in the pathogenesis of diabetes and kidney disease [1012]. While it has been diffi-
cult to demonstrate in vivo activation of the RAS in early or established hypertension
in humans, there is no question that inhibition of the RAS is effective in lowering
62 R.M. Carey
BP in patients with primary hypertension [10]. The results of multiple clinical tri-
als demonstrate that blocking the RAS with angiotensin-converting enzyme (ACE)
inhibitors or angiotensin (AT1 ) receptor blockers (ARBs) not only lowers BP
and BP variability but also reduces cardiovascular events and total morbidity and
mortality [10].
This chapter will focus on new developments in our knowledge of AT1 Rs and the
actions of ARBs in the treatment of hypertension. I will discuss novel mechanisms
regulating AT1 Rs and their actions, review newly discovered RAS pathways with
which ARBs may interact, and interpret recent clinical efficacy studies of ARBs
alone and in combination with other blockers of the RAS.
6.2 Emerging Concepts of AT1 R Regulation and Action
6.2.1 Ang II-Independent Activation of AT1 Rs
Ligand-independent receptor activation has been described for several G protein-

coupled receptors (GPCRs) [13]. Receptors, including AT1 Rs, may exist in two
states, inactive (uncoupled) and active (coupled). Docking of an agonist to the recep-
tor causes more of the receptors to exist in the active state. In the case of the AT1 R,
when the agonist Ang II couples with the receptor, the receptor activates result-
ing in vasoconstriction, aldosterone secretion, sodium and water retention, sympa-
thetic nervous system activation, and increased BP. Even when Ang II is unavailable,
AT1 Rs can still be in the active state. When this occurs, the receptor is said to exhibit
constitutive activity. For example, the native AT1 R can be activated by mechanical
stretching of cardiomyocytes in the absence of Ang II [14].
An inverse agonist is a compound which upon docking to the receptor causes an
increase in the number of receptors to exist in the inactive state (Fig. 6.3). Inverse
agonists may stabilize the inactive conformation of the receptor and drive the equi-
librium away from active conformation. Thus, docking of an inverse agonist reduces
the constitutive (basal) activity of the receptor. The property of inverse agonism is
important because it may help explain the ability of some ARBs to lower BP even
when the levels of Ang II are low, as they are in 25% of patients with primary hyper-
tension [10].
Spontaneous receptor mutations leading to increased constitutive activity (in the
absence of the agonist) have been implicated in human disease, but such mutations
have not yet been described for the AT1 R [15]. Constitutive GPRC activation should
lead to basal G protein and downstream effector activity. Although it is unknown
whether the wild-type AT1 R has constitutive activity in native tissues, the recep-
tor has been shown to have this activity for inositol phosphate (IP) production in
recombinant systems in which basal expression levels are high [16].
A number of ARBs display inverse agonist activity, including olmesartan, EXP-
3174, a metabolite of losartan (but not losartan itself), valsartan, and candesartan
[16]. An extensive evaluation of the inverse agonist properties of olmesartan and its
A B
Autacrine Mechanism Ligand-independent

AT1R activation
Stretch Ang II
Stretch
Mechano-
receptor
AT1R AT1R
Gq Gq
Hypertrophic Hypertrophic
response response
Modified from Yasuda et al.,

Naunyn-Schmeidebergs Arch
Pharmacol, 2007.
Fig. 6.3 Ligand-dependent and ligand-independent activation of AT1 receptors (AT1 R) in car-
diomyocytes leading to hypertrophy. Modified from Yasuda et al., Naunyn-Schmeidebergs Arch
Pharmacol, 2007
molecular mechanisms was published in 2006 [15]. Mutant AT1 Rs with constitutive
activity were expressed in COS-1 cells. Olmesartan suppressed basal IP production
in mutant receptor-transfected cells demonstrating inverse agonist activity. These
studies suggested that the coexistence of carboxyl and hydroxyl groups in the imi-
dazole moiety of olmesartan is essential for potent inverse agonist activity [15].
Site-directed mutagenesis of the transfected AT1 Rs demonstrated that the inverse
agonist activity of olmesartan requires interactions of Lys199 and His256 in receptor
transmembrane VI with the carboxyl group and Tyr113 in transmembrane IV with
the hydroxyl group of the ARB [15].
The possibility of constitutive activation of AT1 Rs in the pathophysiology of
human disease is intriguing. However, at this time more information is necessary
before we can judge the importance of inverse agonists in the RAS and in the phar-
macological therapy of hypertension and related tissue damage.
6.2.2 AT1 R-Activating Autoantibodies
Pre-eclampsia is a condition characterized by hypertension, proteinuria, hypercoag-

ulability, edema, and placental abnormalities. Pre-eclampsia affects approximately
7% of first pregnancies and is one of the leading causes of maternal and fetal
mortality and morbidity worldwide. In addition to the circulating RAS, a separate
64 R.M. Carey
uteroplacental RAS exists in both the mother (decidua) and the fetus (placenta), and
the RAS in the decidua has been found to be activated in pre-eclampsia [17, 18].
Recently, dysregulation of the RAS in the chorionic villi, the tissue responsible for
maternalfetal blood flow, has been described in pre-eclampsia, setting up the pos-
sibility that increased Ang II generated locally may contribute to vasoconstriction
with reduction in maternalfetal exchange of nutrients [19]. In 1999, autoantibodies
that bind to and activate AT1 Rs, termed AT1 agonistic antibodies (AT1 -AAs), were
first described in the circulation of pre-eclamptic women [20]. AT1 -AAs have been
detected only rarely in serum from normal women [20, 21]. Since the initial discov-
ery of AT1 -AAs, multiple groups have confirmed their presence in the circulation
of preeclamptic women and have substantiated that many of the clinical features of
pre-eclampsia may be related to the ability of these autoantibodies to activate AT1 Rs
on a variety of cells [2026]. In addition to pre-eclampsia, AT1 -AAs are present in
renal transplant recipients during an episode of rejection [26, 27].
From studies in experimental models of pre-eclampsia such as the reduced uter-
ine perfusion pressure (RUPP) and double renin/angiotensinogen transgenic rat
models, it is thought that an initiating pathogenic mechanism is decreased blood
flow to the uteroplacental unit. Decreased blood flow results in hypoxia and placen-
tal ischemia, which leads to endovascular injury. The resulting vascular inflamma-
tory response mediated by tumor necrosis factor-alpha (TNF) probably induces
the production of AT1 -AAs which activate AT1 Rs leading to hypertension and
proteinuria.
AT1 -AAs increase the spontaneous beating rate of cultured neonatal cardiomy-
ocytes in an AT1 R-specific manner, and this observation has served as the basis
for AT1 -AA bioassay [20]. AT1 -AAs bind to a seven-amino acid sequence,
AFHYESQ, on the second extracellular loop of the AT1 R (Fig. 6.4). AT1 -AAs
7-aa peptide
(AFHYESQ)
EXTRACELLULAR
PLASMA
MEMBRANE
INTRACELLULAR
Fig. 6.4 Amino acid structure of the rat type-1 angiotensin receptor depicting its seven-
transmembrane domains and extracellular and intracellular termini. AT1 receptor-activating
autoantibodies bind to a seven-amino acid sequence on the second extracellular loop of the receptor
induce extracellular signal-related kinase (ERK) phosphorylation and stimulate

reactive oxygen species generation via NAD(P)H oxidase in similar fashion to Ang
II. Multiple intermediate pathophysiological steps have been stimulated by AT1 -
AA, including increased PAI-1 in trophoblasts and renal mesangial cells, increased
interleukin-6 (IL-6) in mesangial cells, increased Ca++ in AT1 R-transfected Chinese
hamster ovary (CHO) cells, and increased tissue factor in monocytes and vascular
smooth muscle cells [28]. These changes are likely to contribute to the vasoconstric-
tor, inflammatory, and hypercoagulable state in pre-eclampsia.
Currently, there is no specific and effective therapy for pre-eclampsia, which can
lead to premature delivery. ARBs are contraindicated in pregnancy due to the role
of the RAS in fetal tissue development. If maternal circulating AT1 -AAs contribute
to the pathogenesis of pre-eclampsia, as many studies suggest, then blocking the
action of these autoantibodies with a seven-amino acid epitope blocking antibody
may offer the potential for effective treatment.
6.2.3 G Protein Receptor-Interacting Proteins and AT1 Rs
As with most G protein-coupled receptors, AT1 Rs exhibit an endosomal internaliza-

tion/trafficking and intracellular recycling process that governs its activity. Ligand-
dependent AT1 R activation depends upon cell membrane expression, and receptor
internalization is critical for desensitization and inhibition of signal transduction.
Following agonist binding, AT1 Rs undergo rapid endocytosis and downregulation
through -arrestin and dynamin-dependent mechanisms in clatharin-coated vesi-
cles, by receptor phosphorylation, and through interaction with caveolae [29]. The
carboxy-terminal cytoplasmic tail of the AT1 R is involved in the regulation of recep-
tor internalization independently of G protein coupling. Recently, AT1 R internal-
ization has been demonstrated also to be mediated by AT1 R-interacting proteins.
AT1 R-associated protein (ATRAP) is a 17.8-kDa protein with three transmembrane
domains that bind to a 20 amino acid sequence on the intracellular C-terminal
domain of the AT1 R [30] (Fig. 6.5). ATRAP is expressed in many tissues, such as
aorta, heart, liver, and especially kidney where it associates with AT1 Rs in the renal
tubules [31]. ATRAP exerts an inhibitory action on AT1 R signaling by promoting
constitutive internalization of the receptor, thus reducing cell surface receptor num-
ber [32, 33]. Mice with transgenic overexpression of ATRAP exhibit reduced neoin-
timal formation, NAD(P)H oxidase activity, and inflammation in response to injury
compared to their wild-type controls [34]. Furthermore, ATRAP transgenic mice
have smaller cardiac hypertrophic responses to aortic banding than their wild-type
controls, indicating a role for ATRAP in inhibiting cardiovascular remodeling [35].
Recent studies using immunoprecipitation and BRET (bioluminescence reso-
nance energy transfer) microscopy have demonstrated that ATRAP closely asso-
ciates with the C-terminal domain of AT1 Rs, promotes receptor internalization
into cytoplasmic endosomes, and attenuates the Ang II-mediated c-fos/transforming
growth factor- pathway and proliferative responses in vascular smooth muscle cells
[36]. ATRAP is able to interact with AT1 Rs even in the absence of Ang II, but Ang
66 R.M. Carey
AT1-AA
NH2 NH2
Extracellular
Intracellular
COOH COOH
DAG IP3
PKC
Ca++
ERK 1/2
Calcineurin
NAD(P)H
NF- B Oxidase NFAT PAI-1
Fig. 6.5 Schematic illustration of the interaction of AT1 receptor activating autoantibodies (AT1 -
AA) with AT1 receptors and downstream cell signaling mechanisms. DAG diacylglycerol;
PKC protein kinase C; ERK extracellular signal-related kinase; NF-?B nuclear factor kappa
B; IP3; inositol tris-phosphate; Ca++ calcium ion; NFAT Nuclear factor activating T cell; PAI-1
plasminogen activator inhibitor -1
II stimulation significantly facilitates the interaction of ATRAP with AT1 Rs. These
studies have shown that while ATRAP can bind to AT1 Rs under basal conditions,
the major interaction occurs with AT1 Rs that have been internalized into endocytic
vesicles upon Ang II stimulation [36]. Therefore, it is thought that ATRAP may
help keep AT1 Rs internalized even after removal of Ang II. Interestingly, knock-
down of ATRAP increased both basal constitutive and Ang II-mediated AT1 R activ-
ity in these studies [36]. In spontaneously hypertensive rats (SHR), hypertension
was accompanied by a reduction of the ATRAP:AT1 R ratio and cardiac hypertro-
phy [37]. ARB olmesartan recovered the suppressed cardiac ATRAP:AT1R ratio,
decreased AT1 R cell surface density, inhibited p38 mitogen-activated protein kinase,
and reversed the cardiac hypertrophy [37]. One remaining question is whether the
inhibitory mechanism of ATRAP is different from that of ARBs. However, taken
altogether, the results of these studies imply that increased expression/action of
ATRAP, for example, via an activating ligand, could be helpful in reducing detri-
mental AT1 R actions.
Another AT1 R interacting molecule is AT1 R-associated protein (ARAP), a 57.2-
kDa protein which also interacts with the C-terminal tail of the receptor [38]. In
contrast to ATRAP, ARAP promotes recycling of AT1 Rs to the plasma membrane,
suggesting a role in receptor signaling recovery (Fig. 6.6) [39]. ARAP is expressed
mainly in lung, liver, and kidney, but not in the vascular system [40]. Overexpres-
sion of ARAP increases receptor number in the plasma membrane after Ang II stim-
ulation, whereas overexpression of ATRAP does not affect Ang II-mediated AT1 R
NH2
NH2
Extracellular
ARAP ATRAP
Intracellular COOH COOH
NH2
Receptor recycling Receptor
to the plasma Internalization
membrane
Growth Degradation
promotion
COOH signaling
Hypertension
Cardiovascular remodeling
Fig. 6.6 Schematic diagram of the actions of angiotensin receptor-interacting proteins ATRAP
(angiotensin receptor-associated protein) and ARAP (AT1 receptor-associated protein 1) on recep-
tor internalization, sequestration, and recycling to the plasma membrane
internalization. The exact role of ARAP in receptor cycling and its function await
further study.
6.2.4 Receptor Cross-Talk and Dimerization
AT1 Rs have been described to form tightly associated complexes with them-
selves and/or other receptors (Fig. 6.7), including AT2 Rs, mas oncogene recep-
tors, bradykinin B2 receptors, dopamine receptors, endothelin type B receptors,
and epidermal growth factor receptors [4145]. AT2 Rs functionally heterodimerize
cross-talk
Extracellular
AT1R AT1R B2 R AT1R AT1R AT1R AT2R
ETB Intracellular
Mas
Increased AT1R activation Inhibition of AT1R
D1/D2/D3
and signaling signaling
EGF-R
Cell Proliferation and Growth
Fig. 6.7 Schematic representation of cross-talk of AT1 receptors (AT1 R) with other receptors
and the functional actions of homo- and heterodimerization of the AT1 receptor (AT1 R). AT2 R
angiotensin type-2 receptor; B2 R bradykinin B2 receptor; ETB endothelin-B receptor; Mas
mas oncogene receptor; D1 /D2 /D3 dopamine receptors; EGFR epidermal growth factor receptor
68 R.M. Carey
with bradykinin B2 receptors to generate nitric oxide and cyclic GMP [46]. The
functional consequences of AT1 R homodimerization and heterodimerization have
not been fully elucidated. AT2 Rs antagonize the activation of AT1 Rs by direct phys-
ical association [43]. AT1 Rs heterodimerize with bradykinin B2 receptors in pre-
eclampsia, and the receptor complex contributes to Ang II hypersensitivity [44, 45].
The precise role of dimmer formation in the functional activity of AT1 Rs awaits
further study.
6.3 Considerations in the Use of ARBs for Hypertension
6.3.1 Importance of Renal AT1 Rs in the Control of Blood Pressure
The classical RAS has been considered predominantly as a circulating hormonal

system with the enzyme renin, the production of which is supplied exclusively by
renal juxtaglomerular cells, performing catalytic conversion of angiotensinogen to
Ang I, the rate-limiting step in Ang II formation. However, strong evidence now
exists that the RAS also functions as a series of local tissue systems that operate
independently of the systemic circulation [10]. The definition of a tissue hormonal
system requires that all components are present locally and that the final product
(Ang II) is generated locally and engenders a biological action without being trans-
ported outside of the local environment. Local tissue hormonal systems are termed
paracrine (cell-to-different cell), autacrine (cell-to-same cell), or intracrine (intra-
cellular), depending on their relationship to the cell of origin. In 1977, the intrarenal
RAS was first identified as a local cell-to-cell system [47]. Since that time, incon-
trovertible evidence has demonstrated that an independent tissue RAS exists within
the kidney, which is important in the control of fluid and electrolyte balance, kidney
function, and blood pressure [10]. Independent tissue RASs have now been demon-
strated for many other organs including brain, vasculature, heart, adrenal glands,
and the uteroplacental unit, but the evidence for a functionally significant intrarenal
RAS remains the strongest among these (10, 48).
The existence of a functionally significant intrarenal RAS raises important
questions concerning the tissue penetration and distribution of ARBs. Selective
activation of the intrarenal RAS in the renal proximal tubule, independent of
the circulating RAS, has been demonstrated to induce hypertension in renin and
angiotensinogen transgenic animal models [49, 50]. Recently, elegant renal cross-
transplantation studies by Coffman and colleagues [51, 52] have demonstrated that
selective Ang II activation of AT1 Rs in the kidney, particularly in the renal prox-
imal tubules, is required to sustain a hypertensive process. In light of the critical
importance of the intrarenal RAS and renal AT1 R in initiating and sustaining hyper-
tension, the renal distribution of ARBs may be a critical characteristic for effectively
blocking the intrarenal RAS and lowering BP. However, there is very little informa-
tion on the renal partitioning of ARBs; this would seem an important area for future
investigation.
6.3.2 AT2 R Activation in Response to AT1 R Blockade

As discussed above, AT1 R blockade increases renin biosynthesis and secretion by
renal juxtaglomerular cells, leading to increased Ang I and Ang II formation in vivo.
Consequently, Ang II is available to activate unblocked AT2 Rs, which have vasodila-
tor, natriuretic, anti-inflammatory, and antigrowth properties [3]. Indeed, increasing
evidence suggests that AT2 Rs are counter-regulatory to the detrimental actions of
Ang II via AT1 Rs. Recently, Savoia et al. [53] reported that AT2 R expression is
upregulated in the resistance arteries of hypertensive, diabetic patients treated for 1
year with ARB valsartan, but not by -adrenergic receptor blocker atenolol. Resis-
tance arteries from valsartan-treated patients responded to Ang II with vasodilation,
while those from atenolol-treated patients did not [53]. These observations suggest
that chronic AT1 R blockade may improve vasoconstriction and vascular remodel-
ing by upregulation and activation of AT2 Rs and introduce the possibility that non-
peptide AT2 R agonists may be appropriate therapeutic agents for hypertension in
the future.
Other studies have shown that, while AT1 Rs promote sodium retention, AT2 Rs
mediate natriuresis and that des-aspartyl1 -Ang II (Ang III) may be the preferred
endogenous AT2 R agonist for this response [54, 55]. Ang III is a metabolic degrada-
tion product of Ang II via aminopeptidase A (APA) and is itself metabolized to Ang
IV via aminopeptidase N (APN). Inhibiting APN in experimental animals markedly
augments the natriuretic response to exogenous Ang III, indicating that APN inhi-
bition may be an additional therapeutic target in hypertension in the future.
6.3.3 Clinical Efficacy Trials of ARBs in Hypertension
Significant antihypertensive actions and positive clinical outcomes have been

described during treatment with ARBs (losartan, valsartan, eprosartan, irbesartan,
candesartan, telmisartan, and olmesartan). Clinical trials have included patients with
varying degrees of hypertension alone or in combination with other cardiovascular
risk factors, including elderly patients and patients with left ventricular hypertro-
phy, congestive heart failure, post-myocardial infarction, diabetes mellitus, hyper-
lipidemia, resistant hypertension, multiple risk factor combinations, and target organ
damage including renal dysfunction (Table 6.1) [5667].
Characteristics of ARBs that significantly contribute to their high degree of suc-
cess in the treatment of hypertension are (1) placebo-equivalent tolerability, (2) rapid
BP reduction and (3) general 24-hour BP control. Freedom from serious side effects
with ARB therapy renders long-term compliance feasible in the vast majority of
patients. Because BP surges during the early morning hours before awakening, dura-
tion of antihypertensive action of ARBs is paramount.
While ARBs are generally safe and effective, in certain populations caution is
required. Hypotension is sometimes observed in elderly patients and those with
sodium or volume depletion due to activation of the endogenous RAS. ARBs
70 R.M. Carey
Table 6.1 Major angiotensin receptor blocker (ARB) monotherapy clinical efficacy trials
Study Primary end

Clinical trial Study drugs population N Time (y) point
VALUE Valsartan vs HT, high CV 15,245 4.2 Morbidity/

amlodipine Risk mortality
equivalent
SCOPE Candesartan HT, elderly 4,964 3.7 CV death,
vs placebo nonfatal
stroke,
nonfatal MI
equivalent
LIFE Losartan vs HT, LVH 9,193 4.8 Death, MI,
atenolol stroke
reduced
VALIANT Valsartan vs MI, HF, LVD 4,703 2.0 All-cause
captopril vs mortality
comb. equivalent
OPTIMAL Losartan vs AMI, HF, 5,477 2.7 All-cause
captopril LVD mortality
equivalent
CHARM Candesartan HF 7,601 3.0 All-cause
vs placebo mortality
equivalent
VaL-HeFT Valsartan vs HF 5,010 2.3 Mortality
placebo equivalent
ELITE II Losartan vs HF 3,152 1.5 All-cause
captopril mortality
equivalent
INDT Irbesartan vs HT, T2DM 1,715 2.6 Combined
amlodipine nephropa- serum
vs placebo thy creatinine,
ESRD, death
reduced
RENAAL Losartan vs T2DM, 1,513 3.4 Combined
placebo nephropa- serum
thy creatinine,
ESRD, death
reduced
IRMA-2 Irbesartan vs HT, T2DM, 590 2.0 Time to onset
placebo microalbu- nephropathy
minuria reduced
may aggravate hyperkalemia due to their ability to reduce aldosterone secretion.

In patients with bilateral renal artery stenosis, glomerular filtration rate (GFR)
is dependent on Ang II-dependent renal efferent arteriolar constriction. ARB
therapy in this setting can reduce efferent arteriolar resistance, lower GFR, and
induce renal failure [9]. For reasons discussed above, ARBs are contraindicated in
pregnancy.
6.3.4 Pleiotropic Actions of ARBs

Given the myriad of detrimental tissue effects of Ang II summarized in Fig. 6.2,
cardiovascular and renal effects independent of BP have been sought in experi-
mental animal models. Indeed, multiple pleiotropic actions of ARBs as well as
angiotensin converting enzyme (ACE) inhibitors have been described, including
(among many others) (1) blockade of advanced glycation end product-induced
angiogenesis, (2) protection from renal injury in the subtotal nephrectomy model,
(3) protection from renal injury in the metabolic syndrome and diabetes mellitus,
(4) renal protection in uninephrectomized aldosterone/salt-treated rats, (5) protec-
tion from hypertension-induced renal injury, (6) reduction in oxidative stress, (7)
attenuation of cardiopulmonary remodeling and inflammatory signaling in hypoxic
pulmonary hypertension, (8) attenuation of post-myocardial infarction left ventric-
ular remodelling, (9) improvement in cardiac diastolic dysfunction, (10) reversal
of remodeling in atrial fibrillation-induced structural changes in the heart, and (11)
blockade of the decrease in bone mass induced by estrogen deprivation (summarized
in 68, 69). In these animal studies, the beneficial effects were often independent of
the degree of BP control.
Pleiotropic effects of RAS inhibition have been much more difficult to demon-
strate in humans. Whereas relatively small studies with different surrogate end-
points have suggested the likelihood of benefits exceeding those expected from
BP control [69], large clinical trials generally have not confirmed these actions
[68]. In addition, large meta-analyses generally have not lent support to pleiotropic
effects of ARBs. However, there are some notable exceptions: (1) A large meta-
analysis from multiple clinical trials has suggested that RAS inhibition with either
an ACE inhibitor or ARB can reduce the incidence of new-onset diabetes mellitus by
approximately 25 % [69]. (2) In a relatively small but well-controlled study employ-
ing ARB losartan for 1 year in subjects with hypertension demonstrated ameliora-
tion, but not complete reversal, of structural changes due to vascular remodeling
in small resistance arteries as compared to the absence of such improvement with
-adrenergic blocker atenolol, despite a similar degree of BP reduction [70, 71]. In
this study, resistance artery media:lumen ratios were 5.91 in normotensive subjects,
8.34 in hypertensive subjects prior to treatment, 8.32 in hypertensive subjects treated
with atenolol, and significantly reduced to 6.86 in hypertensives treated with losar-
tan [70]. Endothelium-dependent vasodilator responses to acetylcholine also were
improved with losartan but not by atenolol, and endothelium-independent vasodila-
tor responses to sodium nitroprusside were indistinguishable between losartan and
atenolol treatment. These studies suggest a pleiotropic action of ARB treatment
beyond BP control [70, 71]. Larger carefully controlled studies will help clarify
whether there are benefits of ARB treatment independently of BP.
While left ventricular hypertrophy, heart failure, post-myocardial infarction car-
diac remodeling, and renal disease all clearly benefit from RAS blockade, it is dif-
ficult to separate these positive tissue effects from those due to BP reduction. For
a pleiotropic effect of an ARB to be regarded as valid, the effect must be assessed
in the context of time-dependent precision measurements of BP differences. At this
72 R.M. Carey
time, techniques for demonstrating these differences are not sensitive enough to
clearly identify blood pressure-independent effects in large clinical trials.
6.4 Combination Therapy

Combination therapy has been the hallmark of antihypertensive management for
two decades since it has been realized that introduction of medications with dif-
ferent mechanisms of action is more effective than single agent dose-titration [72].
Combinations have included diuretics, RAS inhibitors, calcium channel blockers,
-adrenergic blockers, direct vasodilators, central sympatholytic drugs, and miner-
alocorticoid receptor antagonists. Indeed, JNC VII (2003) provides recommenda-
tions for administration of medications with different mechanisms of action as the
fundamental principle in treatment of hypertension (9).
Recently, it has been hypothesized that combination therapy with ACE inhibitor
and ARB might provide more complete RAS blockade than use of either class
of agent alone. Rationale for combination ACE inhibitor/ARB included possible
increased kinin production, decreased aldosterone secretion, improvement in insulin
sensitivity by different mechanisms, additive effects in heart failure and diabetic
nephropathy, and increased AT2 R activation due to Ang II escape in response to
ACE inhibitors alone via the chymase, tissue plasminogen activator (tPA), and
cathepsin D pathways. Meta-analysis of multiple relatively small studies has sug-
gested advantages of combination ACE inhibitor and ARB therapy over either
agent alone [73]. Combination therapy reduced BP by 4/3 mm Hg when compared
with monotherapy without significant escalation of untoward effects [73]. How-
ever, it could not be determined whether this additive effect on BP resulted from
synergistic interactions of the combination due to differences in individual study
design. Meta-analysis of 35 clinical trials also has suggested that the combination
of ACE inhibitor and ARB is more effective than either alone in the reduction of
proteinuria [74].
The ONTARGET trial program, a large (25,620 patients), randomized clinical
trial quantifying cardiovascular events and total morbidity and mortality published
in 2008, radically changed the approach to ACE inhibitor/ARB combination ther-
apy [75]. The objective of this study was to compare the efficacy of ARB telmisartan
with that of ACE inhibitor ramipril in preventing cardiovascular morbidity and mor-
tality and to determine whether there is any additional benefit if combining telmis-
artan with ramipril compared with ramipril individual therapy. Subjects included
in the ONTARGET trial were 55 years of age and had a high risk of develop-
ing a cardiovascular event with coronary artery disease, peripheral arterial occlusive
disease, cerebrovascular disease, and diabetes mellitus with target organ damage.
The trial was designed to demonstrate non-inferiority of telmisartan compared with
ramipril. BP was significantly lower in the telmisartan group (0.9/0.6 mm Hg greater
reduction) and the combination group (2.4/1.4 mm Hg greater reduction) than in the
ramipril group. In this study, telmisartan and ramipril were equally effective in the
prevention of cardiovascular morbidity and mortality, and combination therapy did
not reduce the primary outcome to a greater extent compared with ramipril alone.
In addition, combination therapy was associated with a higher adverse event rate,
including a significant reduction in glomerular filtration rate, than ACE inhibitor
monotherapy. This study demonstrates that combination therapy with ACE inhibitor
and ARB should not be recommended in high-risk patients.
Another method of combination blockade of the RAS is ACE inhibitor or ARB +
direct renin inhibitor (DRI) therapy. Currently, aliskiren is the only available direct
renin inhibitor available for treatment of humans. Aliskiren blocks the catalytic con-
version of angiotensinogen to Ang I, reducing plasma renin activity and Ang II
formation. Because both ACE inhibition and ARB therapy increase plasma renin
activity, combination with aliskiren seems particularly attractive. Indeed, DRI ther-
apy has been shown to be additive to ACE inhibitor or ARB therapy in lowering BP
in hypertensive patients [76, 77].
Recently, results from the Aliskiren in the Evaluation of Proteinuria in Diabetes
(AVOID) trial have been reported [78]. Patients (599) with hypertension and type-2
diabetes mellitus with nephropathy receiving ARB losartan were studied for their
BP and proteinuric responses to combination with aliskiren. The primary outcome
was urinary microalbumin:creatinine ratio at 6 months of combination therapy.
Aliskiren + losartan induced a small nonsignificant (2/1 mm Hg) further BP reduc-
tion compared with that due to losartan alone. However, aliskiren + losartan induced
a highly significant 20% reduction in urinary albumin:creatinine ratio compared to
losartan alone, suggesting that DRI may have renoprotective effects independently
of BP in this group of patients [78]. Recent studies also show beneficial effects of
aliskiren added to ACE inhibitors or ARBs in heart failure [79]. Taken altogether,
the evidence for combination of ACE inhibitor + DRI or ARB + DRI is incomplete
and requires further study.
6.5 Conclusions
Knowledge of the regulation and actions of AT1 Rs has increased dramatically dur-
ing the past 5 years. We now know that AT1 Rs can be activated independently of
their natural ligand Ang II, but the role of this mechanism in human disease pro-
cesses and of ARBs as inverse agonists thereof remains to be determined. An inter-
esting new mechanism for the pathogenesis of pre-eclampsia is the formation of
activating AT1 R autoantibodies, which probably contribute to hypertension and pro-
teinuria. This discovery opens the door for novel therapeutic approaches, including
the potential use of blocking antibodies. Two major G protein receptor-interacting
proteins have recently been discovered. One of these, ATRAP, appears to play an
important role in AT1 R internalization and desensitization; this suggests the pos-
sibility that an ATRAP-activating ligand could be useful in forcing the AT1 R to
remain in the internalized inactive state. In addition, receptor dimerization to both
itself and to other receptors may play a role in AT1 R activation and downstream
signaling events, but more work needs to be done on the functional consequences of
heter- and homodimerization.
74 R.M. Carey
These and other recent developments in the RAS have introduced several new
considerations in angiotensin receptor blockade for the treatment of hypertension.
We now know that the kidney, and specifically the proximal renal tubule, is criti-
cal for the initiation and maintenance of hypertension. When AT1 Rs are blocked,
renin secretion and Ang II formation are enhanced. Consequently, Ang II is free to
activate unblocked AT2 Rs with potential beneficial effects. One of the major benefi-
cial sites of AT2 R action is the kidney, where Ang III activation induces natriuresis.
Many clinical trials have demonstrated the efficacy and safety of ARB therapy in
hypertension. In spite of a myriad of studies in experimental animals documenting
the beneficial effects of ARB administration on tissue damage in hypertension and
cardiovascular disease, whether pleiotropic actions of ARBs independently of BP
exist in humans is currently a matter of controversy. While there is no question that
RAS blockade reduces BP and conveys benefit in terms of cardiovascular morbidity
and mortality, more complete RAS blockade using a combination of ACE inhibitor
and ARB has not proven beneficial, particularly in a high-risk population. However,
combination of ACE inhibitor or ARB with a DRI shows promise on both BP and
microalbuminuria.
New information from fundamental laboratory studies has successfully informed
the pathogenesis and treatment of hypertension and its cardiovascular and renal con-
sequences during the past 5 years. Continuing basic and clinical investigation should
lead to improved understanding, which will enable better therapeutic approaches in
the future.
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Investigators (2008) Aliskiren combined with losartan in type 2 diabetes and nephropathy. N
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heart failure. Circ Heart Fail 1, 1724.
Chapter 7
Structural and Electrophysiological Remodeling
of the Failing Heart
Walmor C. DeMello
Abstract The influence of the renin angiotensin aldosterone system on the

structural and electrophysiological remodeling of the failing heart is discussed
including changes in cell communication, impulse propagation, and the genera-
tion of cardiac arrhythmias. Particular attention was given to the harmful effects of
angiotensin II and the beneficial influence of ACE inhibitors, AT1 receptor blockers,
and angiotensin [17] on cardiac function and cardiac remodeling. Finally, the role
of the renin angiotensin aldosterone system on the regulation of cell volume in the
failing heart was discussed and evidence was provided that the intracrine RAS plays
an important role in the regulation of cell volume. Pathophysiological implications
of the change in cell volume are discussed.
Keywords Failing heart Renin angiotensin system Structural Electrophysiolo-

gical remodeling Cell volume Intracrine renin angiotensin system
7.1 Morphologic and Functional Abnormalities in the Failing

Heart
Heart failure is a complex pathological process characterized by a decline in con-

tractility involving a cascade of events such as a defect in calcium-handling pro-
teins [1, 2], enhanced oxidative stress [3], apoptosis, and changes in the release of
inflammatory cytokines, endothelin, angiotensin II, and aldosterone [1]. Moreover,
a decreased sarcoplasmic reticulum ATPase protein levels, myofilament dysfunction
[4], a change in orientation of the T tubules toward the longitudinal axis of the fibers
[5], and enhanced microtubule density, which imposes a viscous load on active fila-
ments during contraction [6]. Abnormalities of ion pumps and alteration of hormone
W.C. DeMello (B)

School of Medicine, Medical Sciences Campus, UPR, San Juan, PR, USA
e-mail: walmor.de-mello@upr.edu

82 W.C. DeMello
receptors, fibroblast hyperplasia, recruitment of inflammatory cells are also part of

the complex set of events which culminate in abnormalities of impulse conduction
and arrhythmias [7, 8].
All these events contribute to the reprogramming of the heart muscle at the
molecular and cellular levels. The impairment of electrical synchronization due
to an appreciable reduction of gap junction conductance [9], to a decline of con-
nexin43, which is the main gap junction protein in the heart, and to an abnormal
distribution of this connexin [10, 11] leads to cardiac arrhythmias and to decreased
mechanical efficiency [12]. The mechanism involved in the change of expression
and distribution of Cx43 is not totally clear. However, some factors like angiotensin
II [13] and vascular endothelial growth factor [14] have been suggested. Increasing
evidence has been provided that connexin43 interacts with zona-occludens-1(ZO-1),
that ZO1 promotes the formation and growth of gap junction plaques, and that in
patients with heart failure downregulation of ZO-1 plays an important role in gap
junction formation and stability [11]. Although the measurements of total Cx43 con-
tent, per se, do not provide information on the quantity of open gap junction chan-
nels [15], in the failing heart there is an agreement between immunofluorescence
and electrophysiological studies. The velocity and amplitude of calcium transients
are regulated by phosphorylation of calcium-cycling controllers. The protein kinase
A and the Ca2+ /calmodulin-dependent protein kinases are involved in this process as
well as phosphatases. Increased SR-associated protein phospatase 1 seems involved
in the decline of SR Ca2+ pump activity in the failing heart [16].
7.2 On the Role of the Renin Angiotensin System

The heart is a complex electrochemical and mechanical machine in which the elec-
trical impulse is essential for the generation of contraction. The action potential
which is generated in the sinoatrial node propagates from cell-to-cell through gap
junctions which permit the electrical synchronization of the electrical and mechan-
ical properties of the heart. Therefore, an increase in resistance of the gap junction
reduces the electrical coupling and slows the conduction velocity what facilitates
the generation of re-entrant rhythms. On the other hand, the impulse propagation is
discontinuous due to changes in morphology and variations in gap junctional con-
ductance. This situation is particularly evident under pathological conditions like
heart failure due to the development of interstitial fibrosis, necrosis, and calcifica-
tions.
It is known that the activation of the plasma renin angiotensin system during the
process of heart failure is largely responsible for the impairment of heart function
and remodeling of the ventricle [17, 18] [36].
7.3 On the Harmful Effects of Angiotensin II
It is well known that RAS is activated during the process of heart failure and that it is
responsible for many cardiac abnormalities including impairment of cell communi-
cation and cardiac arrhythmias [12]. The involvement of Ang II on the generation of
7 Structural and Electrophysiological Remodeling of the Failing Heart 83
cardiac arrhythmias is also supported by the finding that studies performed in type
1 knockout mice showed AT1 receptors are involved in the generation of cardiac
arrhythmias [19].
The activation of angiotensin II (Ang II) AT1 receptors is involved in the decline
of gap junction conductance and conduction velocity [20]. Ang II as well as aldos-
terone promote inflammation and apoptosis [1] enhance collagen deposition [12]
with consequent generation of interstitial fibrosis which impairs impulse propaga-
tion throughout the ventricle [21]. The presence of a cardiac renin angiotensin sys-
tem is substantiated by the following findings: (a) the ACE mRNA level is increased
in the failing heart [22]; (b) randomized studies have shown that ACE inhibitors
improve symptoms and survival of patients with heart failure [8] an effect not
necessarily related to the fall in arterial pressure; (c) enalapril increases cell cou-
pling and conduction velocity in the failing heart [23], supporting the view that the
RAS is involved in the generation of re-entrant rhythms including atrial fibrilla-
tion [24]. Ang II enhances the dispersion of repolarization of the action potential
of the failing heart [24] a phenomenon probably related to different expression of
AT1 receptors throughout the ventricle [24]. Because evidence is available that ACE
inhibitors improve sarcoplasmic reticulum (SR) functions and calcium handling in
the failing heart [25], it is possible that abnormalities of the SR functions which are
present in the failing heart including SR Ca leak induced by oxidative stress are, in
part, related to the harmful effects of Ang II (see [26]).
7.4 On the Beneficial Effects of Chronic Blockade of Ang II AT1

Receptors
Measurements of gap junctional conductance performed on cell pairs isolated from

the failing ventricle of cardiomyopathic hamsters showed a large number of cells
weakly coupled. Since Ang II reduces the gap junction conductance in the failing
heart [20], the question whether chronic blockade of AT1-receptors reverses the
decline in cell communication merits serious consideration. Recently, studies have
been performed in cardiomyopathic hamsters treated chronically with losartan for
a period of 3 months, and the results showed that the number of cells with very
low values of junctional conductance (2-8 nS) was significantly reduced by losar-
tan while the group with larger values (18-45 nS) was significantly increased [21].
Moreover, electrophysiological studies performed in the isolated ventricle of ani-
mals treated with losartan indicated a significant decline in the number of fibers
showing nonpropagated action potentials and a higher conduction velocity in lon-
gitudinal and transversal directions. Confocal microscopy performed on the heart
of cardiomyopathic hamsters treated with losartan for 4 months showed a decrease
in percentage area of interstitial fibrosis [21]. It is known that Ang II AT1 receptor
transgene in the mouse myocardium causes myocyte hyperplasia and heart block
[27]. The increased gap junction conductance and impulse propagation found in
animals treated chronically with losartan explains the decline in the incidence of
re-entrant rhythms found in these animals [21]. The improvement of impulse prop-
agation was in part due to the reduction of fibrosis, which is known to be produced
84 W.C. DeMello
by Ang II an effect mediated by AT1-receptor activation and TGF-B1 synthe-

sis [28]. Since interstitial fibrosis causes rupture of cell contacts, it is possible to
assume that the disruption of the interstitial space impairs the flow of current through
gap junctions. The mechanism by which losartan improves cell communication is
probably multifactorial, but mainly related to the prevention of PKC and tyrosine
kinase activation caused by Ang II [29]. Evidence is also available that AT1 block-
ers as well as ACE inhibitors reduce the incidence of atrial fibrillation [30] and
other re-entrant arrhythmias. It is also known that AT1 antagonists restores car-
diac rhyanodine receptor function in the failing heart [26], improving mechanical
performance.
7.5 On the Influence of Extracellular and Intracellular Renin on

Cardiac Function
In nephrectomized animals the level of renin in the heart is extremely low [31], sug-
gesting that cardiac renin is dependent on its uptake from plasma [32]. Two types
of renin receptors have been described: (a) a mannose-6-phosphate receptor, which
is a clearance receptor and binds exclusively to glycosylated form of renin followed
by internalization and degradation without generation of angiotensins [33]; (b) a
specific receptor which is highly expressed in the heart, brain, placenta, and eye
[35]. Experiments performed on myocytes isolated from the rat ventricle indicated
that the nonglycosylated form of renin not only binds to the cell membrane receptor
but is internalized with consequent formation of Ang I and Ang II inside the cell
[34]. These findings open the possibility that intracellular renin has a functional sig-
nificance. Experiments performed in isolated cells from rat ventricle demonstrated
for the first time that intracellular renin reduces cell coupling an effect potenti-
ated by simultaneous intracellular dialysis of angiotensinogen [37]. More recently,
it was found that intracellular administration of renin plus angiotensinogen into car-
diac myocytes isolated from the failing heart enhanced the inward calcium current
an effect abolished by intracellular but no extracellular losartan [38]. These find-
ings lead to the conclusion that renin internalization increases the inward calcium
current through Ang II formation inside the cell of the failing heart. Interestingly,
an intracellular renin receptor has been recently described [38], which when acti-
vated by renin promotes the transport of a transcription factor (PLZF) to the nucleus
with consequent activation of regulatory genes [38]. Moreover, a transcript of renin,
which remains inside the cells, is overexpressed during myocardial infarction [39],
indicating that pathological conditions lead to activation of the intracrine RAS.
7.6 Oxidative Stress, Angiotensin II, and Heart Failure
It is known that Ang II stimulates superoxide production via AT1 receptor and acti-
vation of NADPH oxidase, which is located in many tissues [40]. Enhanced Ang
II levels found during pathological conditions like heart failure and hypertension
causes increased superoxide production, simultaneously with a decrease of NO
activity [4143]. On the other hand, it is known the beneficial effects of AT1
blockers as well as ACE inhibitors in diabetic cardiomyopathy are, in part, related
to the increased availability of NO [44]. Indeed, olmesartan reduced oxidative
stress and hypoxia-induced LV remodeling in part through inhibition of nuclear
factor-KappaB(NF-KappaB) and matrix methaloprotein (MMP-9 activities [45]. In
patients with essential hypertension valsartan decreased QTc-dispersion an effect
probably related to its antioxidative stress effect [46].
7.7 Ang (1-7) Counteracts the Effects of Ang II. But is the
Overexpression of ACE2 Arrhythmogenic?
Previous studies indicated that ACE2 hydrolyze Ang II to Ang (1-7) [47] and
that Ang (1-7) is present in the failing heart [48]. On the other hand, Ang (1-7)
improves the heart function after myocardial infarction in rats [49, 50], leading to
the idea that Ang (1-7) counteracts the harmful effects of Ang II in different sys-
tem including cardiac muscle. Supporting this view is the finding that Ang (1-7) has
antiarrhythmic properties [51]. During myocardial ischemia/reperfusion, Ang (1-7)
reduces the incidence of cardiac arrhythmias due to the activation of the electrogenic
sodium pump and consequent membrane hyperpolarization [52]. Similar effects
have been described in the failing heart [53] in which Ang (1-7) incremented the
pump current an effect suppressed by ouabain. Not only the increase of membrane
potential but also the increment of refractoriness reduced the incidence of cardiac
arrhythmias. Since higher doses of Ang (1-7) incremented the action potential dura-
tion appreciably and generated early-after depolarizations [53], the question remains
if an overexpression of ACE2 with consequent formation of greater amounts of Ang
(1-7) can be arrhythmogenic. This idea is supported by previous observations indi-
cating that in mice overexpressing ACE2, severe cardiac arrhythmias were described
[54]. Considering the high incidence of cardiac arrhythmias patients with heart fail-
ure, it is reasonable to ask which is the meaning of the increased expression in the
failing heart [48]. It is quite possible that other abnormalities like apoptosis, the
release of inflammatory cytokines, as well as the genetic reprogramming of the fail-
ing heart prevent the amelioration of the pathologic process.
7.8 Renin Angiotensin Aldosterone System and Regulation of

Cell Volume. Intracrine Versus Extracellular Renin. An
Integrated Hypothesis
Cell volume activates stretch-sensitive ion channels and is an important contrib-

utor to metabolism, gene expression, and protein synthesis [55, 56]. It is known
that hypotonic stress induced by ischemia, for instance, leads to accumulation of
metabolites intracellularly, with consequent cell swelling due to water entering the
cells. Recent observations [57, 58] indicated that the renin angiotensin aldosterone
system is involved in the regulation of cell volume in normal as well as in the fail-
ing heart. In cells isolated from the failing ventricle and exposed to renin (128 pmol
86 W.C. DeMello
Ang I/ml) plus angiotensinogen (110 pmol Ang I generated by renin by exhaustion),
an increase of cell volume was seen concurrently with the inhibition of the sodium
pump while intracellular administration of renin plus angiotensinogen reduced cell
volume through an activation of the sodium pump [58]. The effect of renin is related
to the formation of Ang II because (a) intracellular Ang II administration activates
the sodium pump an effect abolished by intracellular losartan; (b) ouabain inhib-
ited the effect of both renin and Ang II. The increase of cell volume elicited by
extracellular renin plus Ao is related to the activation of the Na-K-2Cl cotransporter
because bumetanide abolished this effect [58]. On the other hand, since intracellu-
lar Ang II reversed the cell swelling caused by hypotonic solution, it is reasonable
to think that the activation of the intracrine RAS might play a protective role dur-
ing myocardial ischemia by reducing cell volume [58]. These observations seem to
indicate that an important function of the renin angiotensin system is the regulation
of cell volume, with extracellular renin and Ang II increasing the cell volume and
the intracrine RAS reducing it.
The tantalizing importance of these findings to heart cell biology is related to
the finding that alterations of cell volume induce the release of ATP, hormones, and
neurotransmitters as well as elicit the activation of plasma membrane receptors and
integrins which also participate in the regulation of cell volume (Fig. 7.1). Cell vol-
ume regulation following cell swelling involves the efflux of ions through activation
of K+ channels and or anion channels and parallel activation of K+ /H+ exchange and
Cl/HCO3 exchange, while cell shrinkage involves accumulation of ions through dif-
ferent mechanisms including activation of the Na-K-2Cl cotransporter and Na+ /H+
Fig. 7.1 Diagram showing the influence of extracellular Ang II on heart cell volume and the
consequences of cell swelling including the enhanced gene expression and the activation of anionic
channels
exchange [59]. Alteration of cell volume regulation contributes to several diseases

such as diabetic ketoacidosis, liver insufficiency, sickle cell anemia, and infection
[59]. Cell swelling-activated Cl current (ICl swell), which is broadly distributed
throughout the heart, shortens the action potential, depolarizes the cell membrane,
is arrhythmogenic [60], and is also activated by agents that alter membrane tension
and cause mechanical stretch [60].
7.9 Aldosterone and Heart Failure. On the Beneficial Effects of

Eplerenone
Aldosterone receptors are members of a large family of nuclear transcription factors,

which includes thyroxine, glucocorticoids, androgen, estrogen, and progesterone
receptors. Recent studies indicated the presence of aldosterone receptors in the heart
of humans and animals [61]. These receptors are involved in tissue inflammation,
fibrosis, and cardiac hypertrophy [2]. It is known that aldosterone contributes to
cardiac remodeling including fibrosis and LV hypertrophy, congestive heart failure,
and sudden deaths due to ventricular arrhythmias [62]. The Randomised Aldactone
Evaluation Study (RALES), which was conducted with spironolactone in patients at
advanced stages of heart failure, indicated a significant beneficial effect of the drug
on both mortality and morbidity [63, 7]. Other studies indicated that eplerenone is
also of benefit to patients with myocardial infarction and left ventricular dysfunction
[64]. More recent studies indicated that eplerenone reduces cardiac remodeling, par-
ticularly interstitial fibrosis with consequent improvement of impulse propagation
and consequent decline in the incidence of cardiac arrhythmias [65]. The decrease
in the incidence of re-entrant rhythms was related to improvement of impulse
propagation in part due to the decrease of fibrosis but also due to a decrease of
the dispersion of QT interval [65].
7.10 Eplerenone Inhibits the Intracrine Action of Angiotensin II

in the Failing Heart
Recent studies performed on isolated cells from the failing heart showed that chronic
administration of eplerenone, an aldosterone receptor inhibitor, suppressed the
intracrine action of Ang II on peak ICa density while reduced significantly the effect
of extracellular administration of the peptide on inward calcium current [66]. This
is a relevant finding because it might indicate that the mineralocorticoid receptor
(MR) is an essential component and a modulator of the intracrine renin angiotensin
system. MR is an intracellular nuclear receptor that belongs to a large family includ-
ing estrogen, thyroxine, progesterone, and glucocorticoid receptors. The finding that
aldosterone reversed the inhibitory effect of eplerenone on the intracrine action of
Ang II supports the view that aldosterone modulates the intracellular action of Ang
II through the mineralocorticoid receptor [66]. Although the effect of eplerenone
on the intracellular action of Ang II might be multifactorial, evidence was found
88 W.C. DeMello
that the decline in the action of Ang II was related to a decline in the expression of
membrane-bound and intracellular AT1 receptors [66]. Major conclusions of these
findings are as follows: (1) eplerenone inhibits the intracrine action of Ang II; (2)
aldosterone reverses the effect of eplerenone; the mineralocorticoid receptor is a
component of the intracrine RAS; (3) the beneficial effects of eplerenone on cardiac
remodeling are related to the inhibition of the harmful effects of extracellular and
intracellular Ang II.
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Chapter 8
Inhibiting the Renin Angiotensin Aldosterone
System in Patients with Heart Failure
and Myocardial Infarction
Marc A. Pfeffer
Abstract Pharmacologic inhibitors of the renin angiotensin aldosterone system

(RAAS) have played an important role in treating and preventing a variety of cardio-
vascular diseases. Although mainly developed as antihypertensive agents, the life-
saving benefits of inhibiting the RAAS at several steps along its neurohumoral cas-
cade in patients with chronic heart failure and high-risk myocardial infarction have
earned these compounds a central role in the pharmacologic armamentarium. This
chapter will highlight proven accomplishments of various inhibitors of the RAAS
and discuss optimal clinical use of these compounds individually and in combina-
tions in both of these conditions.
Keywords Heart failure Angiotensin-converting enzyme (ACE) Beta-blockers
8.1 Heart Failure
Angiotensin-converting enzyme (ACE) inhibitors earned their well-deserved cor-

nerstone role in the treatment of heart failure from early studies that demonstrated
clear survival benefits. In a small study of patients with severe heart failure and
a life expectancy of less than 1 year, the CONSENSUS trial first highlighted the
importance of inhibiting the renin angiotensin system to prolong survival [1]. This
was followed by the much larger SOLVD program of both symptomatic (treatment)
and asymptomatic (prevention) studies of patients with left ventricular dysfunc-
tion (LVEF less than or equal to 35%) [2, 3]. Randomization to the ACE inhibitor
resulted in a reduction in the risk of death and hospitalization for heart failure among
those who were designated as symptomatic [2]. Those classified as asymptomatic
had a better prognosis and randomization to the ACE inhibitor reduced the rates of
Marc A. Pfeffer (B)

Department of Medicine, Division of Cardiology, Brigham and Womens Hospital, Harvard
Medical School, Boston, MA
e-mail: mpfeffer@rics.bwh.harvard.edu

94 M.A. Pfeffer
hospitalization for heart failure but did not have a significant impact on mortality
[3]. However, with longer follow-up it was possible to demonstrate that this early
benefit eventually became translated into improved survival [4].
The importance of inhibiting the renin angiotensin system in symptomatic heart
failure became further solidified by the V-HeFT II, which directly compared two
active therapy strategies the combination of hydralazine and isosorbide dinitrate
versus the ACE inhibitor enalapril [5]. Although enalapril had been shown previ-
ously to be superior to placebo, this survival benefit even when compared to another
effective strategy underscored the central role of ACE inhibitors in the treatment of
patients with heart failure and reduced left ventricular ejection fraction [5].
8.1.1 Angiotensin Receptor Blockers
With the development of angiotensin receptor blockers (ARB) and the concept of
more complete inhibition by blocking at the angiotensin type I receptor rather than
the ACE inhibitor reduction in the generation of the angiotensin II sparked the hope
that the newer pharmacologic agent would produce even better clinical outcomes.
In the ELITE trial, the first major head-to-head test, the ARB losartan (50 mg daily)
was disappointingly found not even to be as effective as the previously proven dose
of captopril (50 mg three times daily) in patients with symptomatic congestive heart
failure [6]. However, when tested against placebo in heart failure patients that were
previously considered intolerant of an ACE inhibitor, the CHARM Alternative trial
demonstrated the effectiveness of the ARB candesartan in reducing cardiovascular
death and hospitalization for heart failure [7]. The magnitude of the observed risk
reductions was well in the range of what would be anticipated to be achieved with an
ACE inhibitor. Similarly, the small subgroup of patients in Val-HeFT that were not
being treated with an ACE inhibitor appeared to show a large reduction in morbidity
and mortality with the use of the ARB valsartan [8]. Collectively, these findings
support that at proper dosages, an ARB can offer heart failure patients the clinical
benefits achieved with ACE inhibitors.
8.1.2 Aldosterone Antagonist
Since angiotensin II is also an important stimulus for aldosterone release, the

complete neurohormonal profile of RAAS inhibitors would include mineralocor-
ticoid blockers. Spironolactone is an effective diuretic and has been used in the
treatment of hypertension for approximately 40 years. Concerns about producing
clinically important hyperkalemia with combined use of an ACE inhibitor and
a mineralocorticoid blocker initially limited concomitant use. However, RALES
showed that in the context of a carefully monitored clinical trial, an incremental
survival benefit could be achieved in patients with severe heart failure already being
treated with an ACE inhibitor with the addition of spironolactone [9]. Subsequently,
an increase in admissions for hyperkalemia and deaths with non-protocol use of
8 Inhibiting the RAAS in Patients with Heart Failure and Myocardial Infarction 95
spironolactone in the general population underscored that patients participating in

RALES were carefully selected and monitored under a protocol. Diligence must
be exercised when trying to extrapolate the findings from RALES to an even older
population with more severe renal dysfunction than those represented in the clinical
trials [10, 11].
8.1.3 Preserved Left Ventricular Systolic Function Heart Failure
Although approximately one-third to one-half of patients with symptomatic heart

failure are not found to have markedly reduced left ventricular ejection fractions,
these patients have generally not been included in most of the now landmark heart
failure trials. Under names like diastolic heart failure, preserved systolic function
heart failure, heart failure with normal ejection fraction, this poorly characterized
large subset of heart failure patients has only recently been the subject of major
randomized controlled clinical trials. As a consequence, the temporal improvements
in survival achieved in the better studied patients with depressed left ventricular
ejection fraction have not yet occurred in this population [12].
In CHARM Preserved, use of the ARB candesartan did not result in a significant
reduction in the primary end point of cardiovascular death or hospitalization for
heart failure [13]. However, in a secondary analysis, rates of hospitalization for heart
failure were reduced when candesartan was added to conventional therapy. The lack
of benefit of the ARB irbesartan on top of concomitant therapies in an even larger
population of patients with heart failure with preserved systolic function recently
reported in I-PRESERVED provided a very clear message concerning the hazards
of extrapolating results from one population to another [14]. Similarly, a sustained
benefit of an ACE inhibitor was not observed when perindopril was tested in the
preserved left ventricular systolic function heart failure patients in PEP-CHF [15].
Although both ACE inhibitors and ARBs have not been shown to be effective in this
population, TOPCAT, the largest ongoing trial in these patients, is currently testing
whether the use of the aldosterone blocker spironolactone can reduce morbidity and
mortality in patients with symptomatic heart failure and a left ventricular ejection
fraction over 45% [16].
8.1.4 Combining RAAS Inhibitors in the Treatment of Heart

Failure
There was a good deal of enthusiasm for combining the newer ARBs with an ACE
inhibitor with the concept of preserving all the clinical benefits of the proven ACE
inhibitor and adding more by producing more complete inhibition of the adverse
effects of angiotensin II. This concept was particularly attractive since it was known
that angiotensin II can be generated by chymase, which is not under the influence of
converting enzyme, and that chronic ACE inhibitor therapy leads to ACE escape
96 M.A. Pfeffer
where angiotensin II levels creep back up despite continued ACE inhibitor admin-
istration. Val-HeFT was the first major test of this concept in a trial large enough
to assess clinical outcomes. Although no improvement was seen in mortality, the
addition of an ARB to conventional therapy (which for over 90% included an ACE
inhibitor) did result in reductions in hospitalizations for the management of heart
failure [8]. The lack of a survival benefit and the directionally adverse findings in
the large subgroup of patients on beta-blockers dampened the initial enthusiasm for
the clinical use of this combination.
Another major trial in this area, CHARM Added, directly focused on this com-
bination with 100% of its patients on an ACE inhibitor (by design) and 55% treated
with a beta-blocker at baseline [17]. The addition of the ARB candesartan was
shown to reduce cardiovascular mortality and hospital admissions for heart failure.
Both of these components of the primary end point were reduced and these bene-
fits were consistently observed across a wide range of subgroups including different
background doses of ACE inhibitors and importantly in the presence or absence of
beta-blocker use.
8.1.5 Beta-Blockers
This question of whether or not there is an interaction with beta-blockers is par-

ticularly important since the evidence for the use of beta-blockers in patients with
reduced left ventricular heart failure to reduce mortality and hospital admissions for
heart failure was particularly robust. Indeed, three independent major randomized
controlled clinical trials (CIBIS II, COPERNICUS, MERIT-HF) each demonstrated
an approximately 30% reduction in risk of death when a beta-blocker (bisoprolol,
carvedilol and metoprolol, respectively) was added to conventional therapy [1820].
Fortunately, at the time the studies were conducted, ACE inhibitors were already
established as a key therapy for patients with heart failure, and as a consequence
the clear benefits of a beta-blocker were proven on top of an ACE inhibitor. There-
fore, by preserving previously proven advances attributed to the ACE inhibitor and
demonstrating incremental value, the ACE inhibitor beta-blocker combination is
generally considered as the foundation of treatments that should be used together.
This combination is uniformly endorsed by all major heart failure guideline writing
groups [21, 22].
8.1.6 Triple RAAS Inhibitor Combinations

Once an ACE inhibitor and beta-blocker are being used in effective doses, the
information for the third agent for patients with heart failure and reduced left
ventricular ejection fraction is less robust and more opinion than evidence based.
The case for adding mineralocorticoid blockers such as spironolactone is based on
RALES where a clear survival benefit of spironolactone was seen on top of an ACE
inhibitor. However, at the time that study was conducted, the effectiveness of beta-
blockers had not been established and only 11% of these patients were on an ACE
inhibitor beta-blocker regimen at baseline [9]. The case for adding the ARB is
predominantly driven by the CHARM Added experience, which had 55% of its
patients on an ACE inhibitor beta-blocker combination at baseline [17]. Using
subgroups to make clinical decisions is not ideal and this is an area where further
studies are needed. It must be underscored that if a third inhibitor of the RAAS is
employed to attempt to continue to lower cardiovascular risk, then careful monitor-
ing for hyperkalemia, exacerbation of renal dysfunction and symptomatic hypoten-
sion must be conducted to balance risks and benefits [23].
8.2 Myocardial Infarction

The most extensive clinical testing of RAAS inhibitors has been in the management
of patients with myocardial infarction. The rationale for this use stems from animal
studies that demonstrated that ACE inhibitors could attenuate the adverse structural
alterations in left ventricular cavity size and shape produced by an experimental
myocardial infarction, resulting in improved left ventricular function and survival
[24, 25]. Proof-of-concept clinical studies showing attenuation of left ventricular
enlargement with ACE inhibitors led the way for a series of major clinical trials of
patients with recent myocardial infarctions [26, 27].
Survivors of myocardial infarction are at heightened risk for all major adverse
cardiovascular events such as reinfarction, sudden death, developing chronic heart
failure, and stroke. This risk is not uniformly distributed and is related to both
comorbidities as well as the extent of the myocardial infarction and residual left
ventricular dysfunction. Major randomized placebo-controlled clinical trials such
as SAVE, AIRE, TRACE, and SMILE each demonstrated important improvements
in clinical outcomes including survival with the addition of an ACE inhibitor to
patients with a myocardial infarction complicated by either left ventricular dysfunc-
tion and signs of acute pulmonary congestion [2832].
Although survival benefits were also achieved with ACE inhibitors in a broader
acute myocardial infarction population in GISSI-3, ISIS-4, and the Chinese Car-
diac Study Collaborative Group, the absolute and relative increases in survival in
the broader populations were less than those observed in the higher-risk patients
[3335]. The consistency of the improvements in prognosis produced by ACE
inhibitors led to the highest-grade recommendation for their use in patients with
myocardial infarction by international guidelines [36].
8.2.1 Angiotensin Receptor Blockers (ARBs)
With this success of ACE inhibitors, there was a hope, almost an assumption
that ARBs, generally considered to be better tolerated and theoretically viewed as
offering more complete inhibition of the adverse effects of angiotensin II, would
result in even greater improvements in clinical outcomes. As in the heart fail-
ure field, the first major clinical trial of patients experiencing a high-risk myocar-
dial infarction, OPTIMAAL, directly compared the ARB losartan (50 mg) to the
98 M.A. Pfeffer
previously proven dose of captopril from the SAVE trial (target 50 mg three times
daily) [37]. As in ELITE 2, the best outcomes were achieved with the older more
established ACE inhibitor regimen.
Fortunately, this concept of ACE inhibitor versus ARB was further tested in the
VALIANT trial evaluating a different ARB (valsartan, target 160 mg twice daily)
also compared to captopril [38]. In this study, survival and other major cardio-
vascular outcomes including development of heart failure, recurrent myocardial
infarction, and cardiovascular death were all comparable in the ACE inhibitor- and
ARB-treated groups. Using a rigorous prespecified noninferiority analysis, this
study was able to demonstrate that this dose of valsartan preserved the clinical
benefits achieved with the previously proven ACE inhibitor. However, the prestudy
hypothesis of the ARB being superior to the ACE inhibitor had to be rejected.
8.2.2 Combining RAAS Inhibitors in Myocardial Infarction

Treatment: ACE Inhibitor Plus ARB
In addition to directly comparing outcomes between ACE inhibitor- and ARB-

treated patients, VALIANT was also designed to test the combination of the ACE
inhibitor plus the ARB versus the ACE inhibitor alone. Unique to VALIANT was
that it was the first study to compare this combination where the use of the ACE
inhibitor was at the proven dose of a tested agent. Thus, this was the most sci-
entifically rigorous test of the concept of adding an ARB to an ACE inhibitor to
examine whether more complete inhibition of the RAAS would lead to improved
prognosis. Unfortunately, there was no further reduction in risk of cardiovascu-
lar events, and the combination of therapies was less well tolerated than either
monotherapy [38].
8.2.3 RAAS Inhibitor and Beta-Blocker

The early studies demonstrating improvement of outcomes of acute myocardial
infarction patients treated with beta-blockers for the most part excluded the higher-
risk patients with left ventricular dysfunction and/or pulmonary congestion the
group of patients that had the greatest absolute and relative benefits with use of
an ACE inhibitor. Although some patients in the ACE inhibitor trials were being
treated with a beta-blocker and they appeared to experience the same improvements
in outcomes as those not on beta-blockers, this ACE inhibitor beta-blocker com-
bination was not directly examined until the CAPRICORN trial. By adding either
the beta-blocker carvedilol or placebo to patients with acute myocardial infarction
and reduced cardiac function predominantly treated with an ACE inhibitor, the sur-
vival benefit observed in the CAPRICORN trial solidified the importance of the
combination of an ACE inhibitor and beta-blocker in this patient population just as
was established by multiple studies in chronic heart failure [39].
8.2.4 ACE Inhibitor, Beta-Blockers, and Mineralocorticoid Blocker

EPHESUS tested whether the mineralocorticoid inhibitor eplerenone could reduce
the rates of death in a high-risk group of patients with recent myocardial infarction
(those with reduced ejection fraction and at least transient pulmonary congestion)
when added to conventional therapy [40]. The 15% improvement in survival, as well
as important reductions in nonfatal cardiovascular events with the use of eplerenone
on top of a well-managed contemporary regimen with 85% of patients at baseline
being treated with both an ACE inhibitor and a beta-blocker, does indicate that this
triple combination in this cohort of patients with high-risk acute myocardial infarc-
tion offers an advance in medical therapy.
8.2.5 Direct Renin Inhibitor
The development of a direct renin inhibitor, which interrupts the RAAS at the ori-
gin of the angiotensin II-generating cascade, once again offers a promise that will
require rigorous testing to determine its place in clinical care. The initial surro-
gate outcome study in patients with heart failure did show that plasma BNP was
incrementally lowered with the addition of the renin inhibitor aliskiren when added
to background therapy with other RAAS inhibitors [41]. Similarly, early testing
as combination therapy on top of the already proven RAAS inhibitors has com-
menced in a high-risk myocardial infarction population. Whether these preliminary
encouraging findings will lead to a safe and incrementally effective therapy for these
patients remains an open question to be resolved by major morbidity mortality trials.
8.2.6 Safety
Although each unique pharmacologic molecule has a distinctive (usually dose-
related) risk-benefit profile, all inhibitors of RAAS can produce symptomatic
hypotension and exacerbate renal problems. The ACE inhibitors have a higher inci-
dence of angioedema and cough. Fortunately, these patients may better tolerate an
ARB and with the proper dose still derive clinical benefits [7]. Small increases
in creatinine can be anticipated with RAAS inhibition; however, in patients with
marginal renal blood flow supported by the actions of angiotensin II, initiation of
one of these neurohumoral inhibitors can result in a more profound acute reduction
in renal clearances. In these instances, the RAAS inhibitors should be discontinued
and an evaluation for renal vascular disease undertaken. At doses associated with
survival benefits, all inhibitors of RAAS impart a risk of producing life-threatening
hyperkalemia, which must be avoided by monitoring potassium levels. This risk
is not uniform and those with advanced age, chronic kidney disease (particularly
eGFR less than 30 ml per minute), diabetes mellitus, and being treated by combina-
tion RAAS inhibitors are more in jeopardy [42].
100 M.A. Pfeffer
The practice and art of medicine requires judicious use of the best available,
though always incomplete, evidence to make clinical decisions for the individual
patient. Inhibitors of the RAAS are amongst the best studied compounds in cardio-
vascular medicine and offer great deal of data about risks as well as benefits for
skilled practitioners to use to improve the prognosis of their patients.
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50, 19591966.
Chapter 9
Left Ventricular Hypertrophy and Treatment
with Renin Angiotensin System Inhibition
Edward D. Frohlich and Javier Dez
Abstract Hypertensive left ventricular hypertrophy can be considered as the

macroscopic result of the exaggerated growth response of the cardiomyocyte to
the mechanical stress imposed on the left ventricle by the progressively increas-
ing arterial pressure. Besides cardiomyocyte hypertrophy exaggerated cardiomy-
ocyte apoptosis and alterations in the extracellular matrix and the microcirculation
also develop, which lead to the structural remodeling of the myocardium. These
changes may help to explain why left ventricular hypertrophy represents not only
an adaptation to increased pressure load but also an independent risk factor and a
marker of risk of cardiovascular complications in hypertensive patients. Experimen-
tal evidence support the notion that angiotensin II contributes in a significant way to
the development of hypertrophy and remodeling of the hypertensive left ventricle.
In accordance with this, data from a large number of clinical studies have shown
that long-term antihypertensive treatment with drugs inhibiting the renin agiotensin
system, namely angiotensin-converting enzyme inhibitors and angiotensin type 1
receptor antagonists, is associated with regression of left ventricular hypertrophy,
and this is associated with improvement in outcome and with the decrease of the
risk of cardiovascular morbidity and mortality, even independently from changes of
other risk factors, including blood pressure.
9.1 Introduction
Hypertensive heart disease can be defined as the response of the heart to the after-
load imposed on the left ventricle by the progressively increasing arterial pressure
and total peripheral resistance [1]. Hypertensive heart disease is characterized by
increased left ventricular mass (LVM) leading to left ventricular hypertrophy (LVH)
in the absence of aortic stenosis or hypertrophic cardiomyopathy [1].
J. Dez (B)
Centre of Applied Medical Research, University of Navarra, Pamplona, Spain
e-mail: jadimar@unav.es

104 E.D. Frohlich and J. Dez
Hypertension-induced LVH consists of a constellation of molecular and struc-

tural abnormalities of myocardial tissue that result in alterations of LV function,
abnormalities of myocardial perfusion, and disturbances of cardiac rhythm in hyper-
tensive patients [2]. As a consequence, LVH is an independent cardiovascular risk
factor related to cardiovascular complications in these patients. In fact, considered
as a categorical variable, LVH significantly increases the risk of congestive heart
failure, cardiac arrhythmia, and sudden death, as well as of coronary artery disease
and stroke [3]. In addition, when LVM is considered as a continuous variable, a
direct and progressive relationship exists between cardiovascular risk and the abso-
lute amount of LVM. During 4 years of follow-up in the Framingham Heart Study
[4], each 50 g/m2 increase in LVM was associated with a 1.49 increase in rela-
tive risk of cardiovascular disease for men and a 1.57 increase for women. The
effect on cardiovascular mortality was even more striking, with a 1.73 and 2.12
relative risk for each 50 g/m2 for men and women, respectively. More recently, it
has been reported that the continuous relation between LVM and cardiovascular
risk in patients with hypertension remains significant after control for cardiovascu-
lar risk factors, including ambulatory blood pressure [5]. Finally, it has been shown
that the cardiovascular risk decreases significantly in hypertensive patients in whom
LVH regresses with antihypertensive treatment compared to patients in whom LVH
persists and patients who develop de novo LVH in spite of similar hemodynamic
effectiveness of the treatment [6, 7].
Therefore, there is an important need for physicians to understand the patho-
physiology of hypertensive LVH and become fluent in treatment options available.
This chapter details the pathways that are involved in the development of LVH in
hypertension, with a focus on the renin angiotensin system (RAS). Pharmacological
interventions designed to inhibit the RAS and prevent or regress LVH in hyperten-
sive patients are subsequently discussed.
9.2 Role of the RAS in the Development of Hypertensive LVH
9.2.1 General Mechanisms of LVH

The heart is very sensitive to physiological stimuli or pathological states, and even
slight perturbations can lead to severe cardiac changes, eventually with detrimental
outcomes. For instance, in conditions of pressure overload due to systemic hyper-
tension, the left ventricular myocardium undergoes extensive hypertrophy because
of increased size of cardiomyocytes [8]. Functionally, hypertensive LVH is charac-
terized by an initial compensatory process that helps the heart in sustaining cardiac
output despite increased afterload imposed by systemic hypertension [8]. However,
this process is only an initial adaptive response, and chronic exposure to biome-
chanical stress associated with hemodynamic load eventually leads to impaired
inotropic/lusitropic function that, in many cases, progresses to HF [8]. This mal-
adaptive change is accompanied by two types of events that facilitate the transition
from LVH to HF [9]: (i) a switch in the cardiomyocyte gene program that leads to
9 Left Ventricular Hypertrophy and Treatment with RAS Inhibition 105
altered expression of a number of genes, and (ii) changes in the composition of the
myocardium that result in its structural remodeling.
The changes in genetic expression characteristic of the cardiomyocyte hyper-
trophic response involve isogenic shifts, which result in the re-expression of a foetal
gene program, as well as the repression of other post-developmental genes [9, 10].
The long-held views are that these morphological and genetic changes, in response
to pressure overload, serve to restore cardiac muscle economy back to normal, and
counteract myocardial dysfunction. However, there are experiments showing that
a blunting of cardiomyocyte hypertrophy and an attenuation of the foetal gene re-
expression did not result in dysfunction/failure despite pressure overload. Therefore,
a shift in paradigm is occurring in the sense that genetic reprogramming associ-
ated with cardiomyocyte hypertrophy is no longer considered as an adaptive process
[11, 12].
In fact, a detailed analysis of the genetic changes that accompany cardiomyocyte
hypertrophy allows to conclude that they will translate into derangements in energy
metabolism, contractile cycle and excitation-contraction coupling, cytoskeleton and
membrane properties determining mechanical dysfunction, and autocrine functions
which, in turn, will provide the basis for the cardiomyocyte malfunctioning that
is associated with LVH and that predisposes to diastolic and/or systolic dysfunc-
tion [13].
Changes in the composition of cardiac tissue develop in hypertensive LVH
that lead to structural remodeling of the myocardium. Myocardial remodeling
involves increased rates of cardiomyocyte cell death responsible for the decrease in
cardiomyocyte number, exaggerated accumulation of collagen fibers within the
interstitium and surrounding intramural coronary arteries and arterioles leading to
myocardial fibrosis, and structural alterations in the wall of the small intramyocar-
dial vessels that compromises myocardial perfusion (Fig. 9.1). Historically, there
A B
C D
Fig. 9.1 Microscopic view of an apoptotic cardiomyocyte (panel A, arrow), the two patterns
(interstitial, panel B, arrow, and perivascular, panel C, arrow) of collagen deposition leading to
myocardial fibrosis, and an intramyocardial artery presenting wall thickening and lumen narrowing
(panel D, arrow) (right picture) present in a human hypertensive hypertrophied left ventricle (left
picture)
are three types of cell death: apoptosis, autophagy, and necrosis. Although the
three types of cardiomyocyte death have been observed simultaneously enhanced in
human hearts as a consequence of pressure overload or an ischemic insult, apop-
tosis is the most thoroughly characterized form of cardiomyocyte death in the
hypertensive myocardium [14]. Myocardial fibrosis present in LVH is suggested
to be the result of both increased collagen type I and type III synthesis by fibrob-
lasts and phenotypically transformed fibroblast-like cells or myofibroblasts and
unchanged or decreased collagen degradation by matrix metalloproteinases [15].
Hyperplasia and/or hypertrophy and altered alignment of vascular smooth muscle
cells due to changes in extracellular matrix lead to encroachment of the tunica
media into the lumen and cause both an increase in the medial thickness/lumen
ratio and a reduction in the maximal cross-sectional area of intramyocardial
arteries [16].
9.2.2 Role of Angiotensin II in Cardiomyocyte Hypertrophy

and Myocardial Remodeling
Hypertension-induced mechanical stress of cardiomyocytes, cardiac fibroblasts,
and vascular smooth muscle cells elicits paracrine and autocrine signaling by
inducing synthesis and secretion of neurohumoral factors and cytokines that
are responsible for both cardiomyocyte hypertrophy and myocardial remodel-
ing occurring in the hypertensive left ventricle. A number of experimental and
clinical evidence suggest that angiotensin II may contribute to cardiomyocyte
apoptosis [17], myocardial fibrosis [18], and alterations of the wall composi-
tion and geometry of intramyocardial arteries [19] in the hypertrophied left
ventricle.
A recent review of the literature suggests that the involvement of angiotensin II
in the development of LVH is the result not just of its ability to increase blood pres-
sure but mainly of its capacity to alter directly the biophysiology of cardiac cells
[20] (Fig. 9.2). Several experimental and clinical evidences support this notion. On
the one hand, recent studies have demonstrated that salt excess in spontaneously
hypertensive rats (SHRs) produces a modestly increased arterial pressure while pro-
moting marked myocardial fibrosis and structural damage associated with altered
coronary hemodynamics and ventricular function [21, 22]. In addition, the block-
ade of the angiotensin type 1 (AT1 ) receptor performed concomitantly with dietary
salt excess ameliorated salt-related structural and functional cardiac abnormalities
in SHRs without reducing arterial pressure [23]. On the other hand, it has been
reported that old hypertensive patients with high plasma angiotensin II concen-
trations in relation to sodium excretion exhibited a greater LVM, posterior wall
thickness, and septal wall thickness than those with low angiotensin II levels in
relation to sodium excretion, with no differences in blood pressure between the
two groups [24]. In addition, it has been shown that plasma angiotensin II concen-
tration at high sodium intake correlated with LVM independently of ambulatory
ANG IIE
AT AT AT AT
AT11
Cm AT11 Fb AT11 Ec AT11 Vsmc
ANG III
I
Hypertrophy Apoptosis >Collagen <MMP-1 >TIMP-1 Hypertrophy >ECM

Hyperplasia
Alterations of the Alterations of the Alterations of the

cardiomyocyte extracellular intramyocardial
compartment matrix vasculature
Myocardial hypertrophy and remodeling
Fig. 9.2 Schematic representation of the actions of extracellular (ANG IIE ) and intracellular
(ANG III ) angiotensin II in cardiac cells that result in structural alterations of the myocardium
responsible for myocardial hypertrophy and remodeling that characterize hypertensive left ventric-
ular hypertrophy. (Cm, means cardiomyocyte; Fb, fibroblast; Ec, endothelial cell; Vsmc, vascu-
lar smooth muscle cell; AT1 , angiotensin II type 1 receptor; MMP-1, matrix metalloproteinase-1;
TIMP-1 tissue inhibitor of matrix metalloproteinases-1; ECM, extracellular matrix)
blood pressure in young hypertensive individuals [25]. Therefore, locally acting

myocardial angiotensin II can be considered as a major determinant of LVH in
hypertension.
In fact, interacting with its specific G protein-coupled AT1 receptor, angiotensin
II elicits Gaq pathways in the cardiomyocyte that result in activation of several
Ca2+ -dependent signaling molecules (i.e., Ca2+ -dependent protein kinases and
mitogen-activated protein kinases, as well as the Ca2+ -calmodulin-dependent phos-
phatase calcineurin) which, in turn, participate in the transduction of hypertrophic
stimuli to the nuclei [26, 27]. This will activate gene expression and promote protein
synthesis, protein stability, or both, with consequent increases in protein content and
in the size and organization of force-generating units (sarcomeres) that, in turn, will
lead to increased size of individual cardiomyocytes [28]. Of interest, the AT1 recep-
tor itself is directly activated by mechanical stress and invokes the aforementioned
signaling routes that lead to cardiomyocyte hypertrophy, which can be blocked by
an inverse agonist of the receptor [29].
In addition, angiotensin II binding of AT1 receptors triggers apoptosis by a
mechanism involving stimulation of p38 MAP kinase activity, activation of p53
protein and subsequent decrease of the Bcl-2-to-Bax protein ratio, activation of
caspase-3, stimulation of calcium-dependent DNase I, and internucleosomal DNA
fragmentation [30]. Although angiotensin II has been shown to induce apoptosis
in other cardiovascular cells through stimulation of the AT2 receptor, several find-
ings suggest that it is unlikely that this receptor is a strong signal to induce car-
diomyocyte apoptosis in vivo. In fact, apoptosis is not increased in the heart of
transgenic mice overexpressing AT2 receptors in the myocardium [31]. In addition,

Ikeda and colleagues [32] have reported that blockade of AT1 receptors with losar-
tan is accompanied by normalization of cardiac apoptosis in rats with angiotensin
II-induced hypertension that exhibit increased expression of AT2 receptors in the
heart.
On the other hand, increasing evidence supports the notion that angiotensin II
influences both fibrillar collagen synthesis and degradation [33]. In vitro studies of
rat and human cardiac fibroblasts and myofibroblasts have shown that angiotensin
II stimulates cell proliferation and fibrillar collagen synthesis via the AT1 receptor.
The proliferative response of fibroblasts to angiotensin II might well be mediated
by stimulation of the synthesis of growth or inflammatory substances like platelet-
derived growth factor (PDGF) and cytokines, by integrin activation due to secreted
extracellular matrix proteins (e.g., osteopontin), or by a combination of these mech-
anisms [34]. A number of studies provide strong evidence that angiotensin II stim-
ulates collagen synthesis by cardiac fibroblasts via specific growth factors [35]. For
instance, angiotensin II has been shown to induce collagen I gene expression via
activation of both MAP/ER kinase pathway and TGF-1 signaling pathways (e.g.,
connective tissue growth factor - CTGF - and Smad proteins). Finally, angiotensin
II stimulation of the AT1 receptor has been shown to inhibit collagen degradation by
attenuating interstitial matrix metalloproteinase-1 (MMP-1) or collagenase synthe-
sis and secretion in cardiac fibroblasts [36] and by enhancing TIMP-1 production in
rat heart endothelial cells [37].
Studies of human vascular smooth muscle cells and of vessels from experi-
mental animals have demonstrated that angiotensin II binding to the AT1 recep-
tor leads to activation of receptor tyrosine kinases, such as epidermal growthfactor
receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and insulin-
like growth factor-1 receptor (IGF-1R), and nonreceptor tyrosine kinases, such as
c-Src [38]. In addition, AT1 receptor binding by angiotensin II induces activation of
NAD(P)H oxidase resulting in intracellular generation of reactive oxygen species,
which influence redox-sensitive signaling molecules, such as mitogen-activated pro-
tein (MAP) kinase p38MAP kinase, JNK, ERK1/2, and ERK5), and transcription
factors (NF-B, AP-1, and hypoxia-inducible factor-1) [39]. These signaling events
stimulate vascular smooth muscle cell growth and extracellular matrix production.
Some of the cardiac effects of angiotensin II seem to be independent of the AT1
receptor but the result of the ability of the peptide to operate in the intracellular
space. As early as 1971, it was reported that tritiated angiotensin II is internalized
and rapidly localized to the nuclei and mitochondria of cardiomyocytes [40]. Subse-
quently, Re and colleagues [41] demonstrated the presence of specific, high-affinity
nuclear receptors for angiotensin and showed that nuclear binding of angiotensin II
enhanced gene transcription. In 2004, Baker and colleagues [42] demonstrated that
the transfection of cardiac myocytes with a construct encoding a nonsecreted type
of angiotensin II led to the rapid induction of cell hypertrophy. In fact, when a plas-
mid encoding the nonsecreted angiotensin II, the expression of which was driven by
the -myosin heavy chain promoter, was injected into mice, marked LVH developed
within 96 h [42].
9.3 Effects of Pharmacological Inhibition of the RAS on

Hypertensive LVH
9.3.1 General Aspects
Antihypertensive drugs are effective in reducing LVM. In fact, Mosterd and col-
leagues [43] analyzed recently the data from 10333 participants in the Framing-
ham Heart Study, and reported that the increasing use of effective antihyperten-
sive therapy has caused a decrease in the prevalence of both hypertension and
LVH in the general population. A large number of trials and meta-analyses have
attempted to compare the effects of different antihypertensive agents on LVM,
but flawed study designs and methodological problems have limited the utility of
these studies. Nevertheless, a recent meta-analysis by Klingbeil and colleagues
[44] including 80 double-blind, randomized controlled trials with 146 active treat-
ment arms (n = 3767 patients) and 17 placebo arms (n = 346 patients) showed
that after adjustment for treatment duration and change in diastolic blood pressure,
there was a significant difference among medication classes in regressing LVH. In
fact, the decrease in LVM (indexed by body surface area or LVM index) induced
by the different classes was as follows: AT1 receptor antagonists>calcium channel
blockers>angiotensin-converting enzyme (ACE) inhibitors>diuretics>beta-blockers
(Fig. 9.3). In pair-wise comparisons, AT1 receptor antagonists, ACE inhibitors, and
calcium channel blockers were more effective at reducing LVMI than were diuretics
and beta-blockers.
Why might AT1 receptor antagonists and ACE inhibitors decrease LVMI more
effectively than would the other antihypertensive agents? The findings from the
meta-analysis by Klingbeil and colleagues [44] indicate that blockade of the RAS
reduces LVM beyond the effects of blood pressure reduction. This has been clearly
demonstrated in two studies. A substudy of the HOPE trial showed that compared
with patients given placebo, ramipril-treated patients without LVH at baseline had
lower rates of subsequent LVH [45]. In addition, ramipril-treated patients with
LVH at baseline had higher rates of regression of LVH, which was associated with
20
Reduction of LVMI (%)
16
12 ARAs
CCBs ACEIs
8
Ds
BBs
4
Fig. 9.3 Reduction (expressed as 95% confidence interval) of the left ventricular mass index
(LVMI) by different classes of antihypertensive drugs (ARAs, angiotensin receptor antagonists;
CCBs, calcium channel blockers; ACEIs, angiotensin converting enzyme inhibitors; Ds, diuretics;
BBs, beta-blockers) (Adapted from [44])
improved prognosis and was independent of blood pressure reduction. Devereux and
colleagues [46] conducted an echocardiographic substudy of the LIFE trial, in which
LVM was measured yearly up to 5 years. Patients randomly assigned to receive
losartan-based therapy had a significantly greater reduction in LVMI compared with
patients receiving atenolol-based therapy. The larger relative reduction in LVMI in
the losartan group was detected after 1 year of treatment and persisted to year 5.
Consistent with the clinical findings of the LIFE trial, regression of LVH was greater
in losartan-treated patients than in atenolol-treated patients even though blood pres-
sure reductions were similar for the two groups. Therefore, the high effectiveness of
pharmacological inhibition of the RAS to reduce LVM and regress LVH seems to be
related to the reduction of the direct pro-hypertrophic and pro-remodeling actions
of angiotensin II on the myocardium.
9.3.2 Emerging Clinical Aspects

The time has come to revisit the current management of hypertensive LVH simply
focused on controlling blood pressure and reducing LVM. In fact, it is necessary to
pay attention also to the correction of alterations in LV function and coronary micro-
circulation that associate with LVH. Although several trials have been performed
to analyze these aspects, methodological problems of design and the confound-
ing influence of factors such as the antihypertensive and antihypertrophic effects
of treatment make difficult to evaluate the available information. Nevertheless, from
the available data it has been proposed that the use of either ACE inhibitors or AT1
receptor antagonists provides a higher benefit than the use of other agents [47, 48].
Similarly, it is now the time to develop new approaches in the treatment of hyper-
tensive LVH aimed to repair myocardial structure (i.e., cardiomyocyte apoptosis
and myocardial fibrosis). The in vivo effects of antihypertensive drugs on cardiac
apoptosis in SHRs have been reviewed elsewhere [49]. Collectively, the available
findings suggest that the ability of antihypertensive drugs to inhibit cardiomyocyte
apoptosis is independent of their antihypertensive efficacy, but can be related to
their capacity to interfere with the pro-apoptotic actions of humoral factors such as
angiotensin II. This is further supported by clinical findings showing that despite
an identical antihypertensive efficacy, the AT1 receptor antagonist losartan, but not
the calcium channel blocker amlodipine, reduced cardiomyocyte apoptosis in hyper-
tensive patients with LVH after 1 year of treatment [50] (Table 9.1). Brilla and col-
leagues [51] showed that treatment with the ACE inhibitor lisinopril, but not with the
diuretic hydrochlorotiazide, reduced myocardial fibrosis in hypertensive patients,
independently from blood pressure control and LVH regression, and that this was
associated with improved left ventricular diastolic function. We have shown recently
that treatment with the AT1 receptor antagonist losartan was associated with inhi-
bition of collagen type I synthesis and regression of myocardial fibrosis in hyper-
tensive patients with LVH [52] (Table 9.1). In contrast, hypertensive patients treated
with the calcium-channel blocker amlodipine did not show significant changes in
collagen type I metabolism or myocardial fibrosis [52] (Table 9.1). Interestingly,
Table 9.1 Effect of antihypertensive treatment on hemodynamic, left ventricular mass and
myocardial histological parameters in hypertensive patients
Losartan group Amlodipine group
At baseline After treatment At baseline After treatment
Study 1
N 21 16
Systolic BP (mm Hg) 1736 1372 16211 1374
Diastolic BP (mm Hg) 952 812 953 792
LVMI (g/m2 ) 1316 1054 13412 11911
CVF (%) 5.650.44 3.960.32 4.930.27 4.310.38
Study 2
N 14 14
Systolic BP (mm Hg) 1732 1362 1767 1393
Diastolic BP (mm Hg) 943 783 963 822
LVMI (g/m2 ) 1349 1056 12713 12411
AI (TUNEL positive 2843730 1118176 1658244 3211639
nuclei/106 nuclei)
Study 1 adapted from [52]. Study 2 adapted from [50].

N means number of subjects in each group. BP means blood pressure. LVMI means left
ventricular mass index.
CVF means myocardial collagen volume fraction. AI means cardiomyocyte apoptotic index.
Data are expressed as meanSEM. P<0.05 vs values at baseline. P<0.01 vs values at
baseline.
the effect of the two compounds on blood pressure was similar all along the treat-
ment period. We have reported also that the ability of losartan to induce regression
of severe myocardial fibrosis is independent of its capacity to reduce blood pressure
or LVM, but it is associated with a diminution of myocardial stiffness in hyper-
tensive patients with LVH [53]. These data support experimental studies in SHRs
where pharmacological interference with the production and actions of angiotensin
II has proved to be effective on reversing cardiac fibrosis beyond the antihyperten-
sive efficacy [54-56].
9.3.3 Emerging Pharmacological Aspects

Although targeting the ACE and the AT1 receptor initially inhibits the RAS, the
Randomized Evaluation of Strategies for Left Ventricular Dysfunction (RESOLVD)
trial clearly demonstrated the concept of aldosterone escape. The authors studied
a cohort of 768 patients with a depressed ejection fraction (<40%) and New York
Heart Association II-IV class heart failure treated with candesartan, enalapril, or
both for 43 weeks. At 17 weeks there was a significant reduction in aldosterone
levels from baseline, but by 43 weeks this improvement disappeared [57]. Mecha-
nisms of aldosterone escape are speculative and include ACTH as a secretagogue
and chymase generation of angiotensin II by a non-ACE inhibitor pathway [58].
Abundant experimental evidence suggests that aldosterone may facilitate LV

growth and myocardial remodeling, beyond the ability of the hormone to facilitate
sodium retention and increase blood pressure, involving the activation of miner-
alocorticod receptor-dependent pathways in cardiac cells that result in hypertrophy
and fibrosis [59]. Recent clinical data by Muiesan and colleagues [60] add strong
support to this notion. The authors evaluated the appropriateness of observed LVM
to the theoretical value of LVM predicted by sex, body size, and stroke work in
125 patients with a diagnosis of primary aldosteronism (PA) and in 125 age-, sex-,
and blood pressure-matched patients with essential hypertension. The prevalence
of inappropriate LVM (defined by a ratio of observed:predicted LVM >135%) was
greater in PA patients than in essential hypertensive patients, irrespective of the
presence or absence of traditionally defined LVH. In addition, direct correlations
were observed between the ratio of observed:predicted LVM and the ratio of aldos-
terone:renin or the post-saline infusion aldosterone concentration in PA patients.
Thus, an excess of aldosterone may induce an additional increase of LVM beyond
hemodynamic load.
In this conceptual framework, recent data suggest the benefit of aldosterone
receptor blockade in addition to the other anti-RAS therapies. In fact, Pitt et al.
[61] studied 202 hypertensive patients with LVH treated for 9 months with the
aldosterone blocker eplerenone, the ACE inhibitor enalapril, or both. LVM was
determined by cardiac magnetic resonance imaging. Despite all groups achieving
the same degree of blood pressure lowering, the combination group had the largest
degree of LVH reversal.
Blockade of the RAS leads to a feedback increase in renin synthesis and secre-
tion. In the case of ACE inhibitors, large increases in the activity of renin in plasma
(referred to as PRA) and angiotensin I can be observed [62, 63]. With the AT1 recep-
tor antagonists, increases in PRA and angiotensin II are observed [64, 65]. Diuretics
also enhance PRA [66]. As reviewed recently [67], several studies show an associa-
tion between PRA and LVM in hypertensive patients. Therefore, it seems reasonable
to propose that more complete RAS inhibition may afford greater protection from
LVH.
Aliskiren is the first orally active direct renin inhibitor to be approved for
the treatment of arterial hypertension. Clinical studies in hypertensive patients
have shown that aliskiren is effective and safe in lowering blood pressure [68].
Concerning the mode of action, direct renin inhibition decreases PRA and
inhibits the formation of angiotensin I and II and most likely also angiotensin
peptides [69].
There is scarce experimental information concerning the impact of direct renin
inhibition on hypertensive LVH. Recently, the effects of 9 months of treatment with
aliskiren, losartan, and their combination were compared on the reduction of LVMI
in hypertensive patients with increased LV wall thickness, and body mass index >25
kg/m2 . LVMI was reduced significantly from baseline in all treatment groups. The
reduction in LVMI in the combination group was not significantly different from
that with losartan alone. Aliskiren was as effective as losartan in reducing LVMI.
Therefore, aliskiren was as effective as losartan in promoting LVH regression [70].
Recently, it has been reported that aliskiren suppresses PRA in combination with
a thiazide diuretic, an ACE inhibitor, or an AT1 receptor antagonist in hypertensive
patients [71]. Thus, renin inhibition offers the prospect of optimize PRA suppression
in patients with hypertension. It remains to be seen whether this effect will translate
in greater cardiac protection. This aspect requires specific investigation taking into
account that with aliskiren, the increase in renin secretion and circulating renin con-
centration are greater than with an ACE inhibitor or an AT1 receptor antagonist [72].
High extracellular levels of renin could conceivably interact with the prorenin/renin
receptor located at the plasma membrane level [73] and as a consequence activate
cardiac fibrotic signaling pathways independent of angiotensin II [74, 75]. In addi-
tion, it must be considered the possibility that intracellular renin originated from
extracellular renin internalization, a nonsecreted isoform of renin or the enhanced
expression of the renin gene elicited by different pathological conditions involving
stretch may also interact with the perinuclear prorenin/renin receptor [76] reducing
cell communication in the heart [77].
9.3.4 Emerging Molecular Aspects

The knowledge of polymorphisms in genes that potentially influence pharmacody-
namic mechanisms would allow the identification of individuals who are likely to
have beneficial responses to treatment with a particular drug. LVM is a complex
phenotype influenced by the interacting effects of multiple genetic and environmen-
tal factors. Genetic variation probably contributes to inter-individual differences in
the LVM by virtue of effects on blood pressure level as well as via pathways that
are not captured by measurements of blood pressure. It is possible that identifica-
tion of genes that influence LVM may enhance the ability to detect those patients
that deserve early treatment to prevent the development of LVH. In this regard, a
recent meta-analysis of case-control studies and association studies has shown that
the D allele of the insertion/deletion (I/D) polymorphism of the ACE gene behaved
as a marker for LVH in untreated hypertensive patients [78], thus making patients
with the DD genotype susceptible to be treated with either ACE inhibitors or AT1
receptor antagonists to prevent LVH.
Based on evidence suggesting that angiotensin II may participate in the devel-
opment of myocardial fibrosis in hypertensive LVH via activation of AT1 recep-
tors, we investigated the potential role of the A1166C polymorphism of the AT1
receptor gene in predicting the antifibrotic effect of antihypertensive drugs [79].
The antifibrotic effect was assessed by measurement of the serum concentration
of the carboxy-terminal propeptide of procollagen type I or PICP, a peptide that
is cleaved from procollagen type I during the synthesis of fibril-forming collagen
type I and that has been shown to be associated with the volume of myocardial
tissue occupied by collagen fibers in the human hypertensive heart [80]. In the
study, hypertensive patients with LVH were studied before and after 1-year treat-
ment with the AT1 receptor antagonist losartan or the beta-blocker atenolol. Base-
line PICP was significantly increased in AA hypertensives compared with AC/CC
hypertensives. Confounding factors were similar in the two subgroups of hyperten-

sives. Administration of losartan was associated with significant reduction in PICP
in AA hypertensives but not AC/CC hypertensives. Treatment with atenolol did not
change PICP in either subgroup of hypertensives. Blood pressure was reduced to
the same extent in the four treatment subgroups. If these results were confirmed
by larger, prospective, double-blind studies, the genotype of the A1166C polymor-
phism of the AT1 receptor gene could be a useful indicator for antihypertensive drug
strategy aimed to reduce myocardial fibrosis in hypertensive patients with LVH.
9.4 Concluding Remarks
Hypertensive LVH is the response of cardiac muscle cells to increased hemody-

namic load. The increase in ventricular wall thickness normalizes increased wall
stress and, therefore, LVH is initially beneficial. However, sustained and progres-
sive mechanical load is associated with additional responses of the cardiomyocyte
and noncardiomyocyte compartments of the myocardium that result in its struc-
tural remodeling and the deleterious long-term consequences on myocardial func-
tion, electrical activity, and perfusion that significantly increase the risk of mor-
tality in hypertensive patients with LVH. Angiotensin II plays a major role in the
detrimental response of the myocardium to chronic mechanical overload imposed
by systemic hypertension on the left ventricle. Therefore, treatment of hyperten-
sive patients with LVH with drugs inhibiting the RAS will take advantage of its
beneficial features while removing the deleterious impact of angiotensin II on the
myocardium. Whereas ACEIs and ARAs have proven their efficacy in regressing
LVH and reducing the associated risk in hypertensive patients, more studies are
necessary to ascertain whether aldosterone blockers and renin inhibitors also pos-
sess cardioprotective properties in terms of LVH regression.
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Chapter 10
Angiotensin-(1-7), Angiotensin-Converting
Enzyme 2, and New Components of the Renin
Angiotensin System
Aaron J. Trask, Jasmina Varagic, Sarfaraz Ahmad, and Carlos M. Ferrario
Abstract The discovery of angiotensin-(1-7) [Ang-(1-7)] in 1988 represented

the first deviation from the traditional biochemical cascade of forming bioactive
angiotensin peptides. Prior to that time, the biological actions of angiotensin II
(Ang II) were being investigated as it relates to cardiovascular function, includ-
ing hypertension, cardiac hypertrophy and failure, as well as biological actions in
the brain and kidney. We now know that Ang II elicits a whole host of actions
both within and outside of the cardiovascular system. Furthermore, the discov-
ery of Ang-(1-7) by our laboratory was also the first indication of a biologically
active angiotensin peptide that further studies revealed served to counter-balance
the actions of Ang II. This chapter reviews the data demonstrating the role of the
vasodepressor axis of the renin angiotensin system in the regulation of cardiovas-
cular function and the new data that shows the existence of angiotensin-(1-12) as
a novel alternate substrate for the production of angiotensin peptides. The ultimate
role of this discovery, as well as the continuing elucidation of mechanisms pertain-
ing to RAS physiology, will likely be clarified in the coming years, in hopes of
improving the treatment of cardiovascular disease.
Keywords Angiotensin-(1-7) Angiotensin-(1-12) Angiotensin II

Hypertension Mas receptor Blood pressure regulation
10.1 Introduction
The existence of the renin angiotensin system (RAS) as a major physiological reg-
ulator has been known since Tigerstedt and Bergman first discovered the enzyme
renin over a century ago [1]. Nearly 60 years after the initial discovery of renin,
A.J. Trask (B)

Hypertension and Vascular Research Center, Department of Physiology & Pharmacology, Wake
Forest University School of Medicine, Winston-Salem, NC
e-mail: aaron.trask@nationwidechildrens.org

122 A.J. Trask et al.
Irvine Page from the United States and Braun Menendez from Argentina indepen-
dently discovered a pressor hormone, angiotonin or hypertensin, which was
later agreeably called angiotensin (Ang). We now know this hormone to be the
octapeptide pressor hormone, Ang II, which is produced from the sequential cleav-
age of the protein (Aogen) into Ang I by renin and Ang I into Ang II by angiotensin-
converting enzyme (ACE).
This linear hydrolysis cascade was undisputed for many years until studies from
our laboratory in 1988 showed that a previously considered inert metabolite of
Ang II, Ang-(1-7), caused the release of vasopressin from the rat brain hypotha-
lamus [2]. This study was the first demonstration of biological activity of a pep-
tide within the RAS that was not Ang II-mediated. The discovery of Ang-(1-7)
expanded our knowledge about the complexities of the RAS and has garnered
increasing support for a potential target for the therapeutic treatment of diseases
such as hypertension, heart disease, and even cancer [3, 4]. This chapter focuses
on the functional role of Ang-(1-7) in the heart, as well as the important contribu-
tion that angiotensin-converting enzyme 2 (ACE2) plays in degrading Ang II into
Ang-(1-7). While the evidence for a protective role for this counterbalancing arm
of the RAS continues to accumulate, we also comment on the identification of a
new angiotensin peptide upstream of Ang I, called angiotensin-(1-12), and how it
may function in tissues as an alternate precursor for angiotensin peptide produc-
tion. A diagram of the most current view of the renin angiotensin system is shown
in Fig. 10.1.
Fig. 10.1 Current view of the renin angiotensin system. Abbreviations: ADAMs, tumor necrosis
factor- convertases, such as ADAM17; sACE2, secreted ACE2
10 Ang-(1-7) and ACE2 123
10.2 Angiotensin-(1-7): Gaining Favor in the 21st Century

The discovery of Ang-(1-7) in the late 1980s did not lend itself to ready acceptance
[2, 57]. However, studies investigating the role of Ang-(1-7) are on the rise, and a
whole new array of data has been emerging on this bioactive peptide since the turn
of the 21st century. Most of the biological effects of Ang-(1-7) that are discussed
below have been attributed to the mas receptor, which was identified as a functional
receptor for Ang-(1-7) [8].
10.2.1 Angiotensin-(1-7) and the Regulation of Cardiac Dynamics
The presence of Ang-(1-7) in the heart and the ability of the heart to produce Ang-
(1-7) were not known for some time. Ang-(1-7) was identified in cardiomyocytes
of the heart, but not cardiac fibroblasts, and Averill et al. [9] further showed that its
expression was augmented after coronary artery ligation. Several studies have shown
that Ang-(1-7) can be synthesized by the heart, and we showed a direct conversion
of Ang II into Ang-(1-7) in isolated hearts from normal and hypertensive rats [10].
Early studies investigating a direct role for Ang-(1-7) actions in the heart showed
that it was protective against ischemia-induced cardiac dysfunction [1113], which
may be due in part to the activation of the sodium pump [14]. Additional studies in
the cardiomyopathy hamster showed that the anti-arrhythmic effects of Ang-(1-7)
are mediated through hyperpolarization of the heart cell [15]. Further evidence that
Ang-(1-7) is a direct positive effector in the heart stems from data showing its anti-
fibrotic and anti-hypertrophic actions [1620]. Because Ang-(1-7) is a peptide and
thus has a short half-life, several studies have investigated a more stable analog of
Ang-(1-7), called AVE0991. The administration of this Ang-(1-7) analog is associ-
ated with improvement of cardiac function in diabetic rats [21], improved barore-
ceptor sensitivity [22], and potentiation of the vasodilator actions of bradykinin [23].
The mas receptor mediates the signaling mechanisms produced by Ang-(1-7)
[8]. We further showed that transfection of cultured myocytes with an antisense
oligonucleotide to the mas receptor blocked the Ang-(1-7)-mediated inhibition of
serum-stimulated mitogen-activated protein kinase (MAPK) activation, whereas a
sense oligonucleotide was ineffective [19]. In keeping with these findings, chronic
mas deficiency leads to impaired Ca2+ handling in cardiomyocytes in culture [24].
10.2.2 Salt and the ACE2/Ang-(1-7)/mas Axis
A clear relationship between salt intake and prevalence of hypertension has been
shown in abundant epidemiological and interventional studies [2528], giving sup-
port for the current recommendation for sodium intake of 2,400 mg per day by
the American Heart Association. On the other hand, despite numerous studies
suggesting that interruption of ACE2/Ang-(1-7)/mas receptor axis may lead to
hypertension and cardiac dysfunction [2931], little is known about its response to
altered sodium intake with respect to blood pressure changes or target organ dam-
age. Our laboratory was among the first one to report the importance of a tonic
depressor activity of Ang-(1-7) to the maintenance of blood pressure in the spon-
taneously hypertensive rats, with endogenous RAS activation induced by chronic
salt depletion [32]. Furthermore, in the face of unchanged plasma Ang-(1-7), an
enhanced vascular sensitivity to endogenous Ang-(1-7) in salt-restricted state sug-
gests significant amplification in Ang-(1-7) receptorsignaling interaction. Subse-
quent studies also revealed that, under the condition of increased renal Ang II due
to salt depletion [33] or 2K1C Goldblatt hypertension [34], endogenous Ang-(1-7)
counterbalanced the effects of Ang II to maintain a glomerular filtration rate and
renal plasma flow [34]. Thus, could it be possible that insufficient synthesis or
activity of ACE2/Ang-(1-7)/mas may be a critically important mechanism in salt-
sensitive hypertension?
Indeed, it has been shown that in female Dahl salt-sensitive rats fed high-
salt diet, chronic Ang-(1-7) supplementation reduced increase in blood pres-
sure and improved aortic and renal blood flow by increasing prostacyclin and
prostaglandinE2 release. It was believed that an increase in plasma levels of nitric
oxide following Ang-(1-7) infusion was responsible for this observed vasodilatory
effect [35]. It has also been shown that acute vasodilation by Ang-(1-7) was aug-
mented in rats fed high-sodium versus low-salt diet due to an increase in vasodila-
tory and a decrease in vasoconstrictor prostanoids [36]. But the antagonistic and
nitric oxide-independent effect of Ang-(1-7) on Ang II-induced vasoconstriction in
aortic rings from the rats fed high-sodium diet was abolished in rats fed low-sodium
diet [37]. Thus, further studies are warranted to define precisely a fine-tuning mech-
anism of Ang-(1-7) in the regulation of blood pressure and flow as well as vascular
reactivity in different status of sodium intake. In this context, it is important to note
that in salt-sensitive hypertensive patients, omapatrilat, a dual ACE and neprilysin
inhibitor, effectively reduced blood pressure and increased urinary excretion of Ang-
(1-7) [38]. This study clearly pointed out that besides the inhibition of Ang II pro-
duction and degradation of atrial natriuretic peptide and bradykinin, an Ang-(1-7) of
renal origin may contribute to the hypotensive effect of omapatrilat in the patients
whose blood pressure is sensitive to sodium intake.
Finally, having in mind the anti-hypertrophic and anti-fibrotic effects of Ang-
(1-7), it is intriguing to hypothesize that salt-induced left ventricular remodeling
and renal injury observed in different forms of experimental and human hyperten-
sion [3943] may be, at least in part, governed by alteration of the ACE2/Ang-(1-
7)/ mas axis. In fact, in Dahl salt-sensitive rats fed high-salt diet, cardiac enlarge-
ment and fibrosis were associated with an increased cardiac angiotensinogen but
reduced cardiac ACE2 mRNA. Treatment with AT1 receptor antagonist, but not
mineralocorticoid receptor blocker, reversed the effect of salt on ACE2 gene expres-
sion [44]. Importantly, both therapies ameliorated salt-induced cardiac remodel-
ing along with a reduction in angiotensinogen and ACE mRNA. Therefore, it
seems that the effects of salt-intake variation or RAS blockade may ultimately
depend on their net effects on the two opposing arms of the RAS. Furthermore,
low sodium intake in Wistar rats reduced renal ACE, but not ACE2 mRNA and
10 Ang-(1-7) and ACE2 125
activity; this effect was not amplified during ACE inhibition [45]. Neither plasma
Ang II nor Ang-(1-7) were affected by low sodium intake, but ACE inhibition
increased plasma Ang-(1-7) shifting the balance between the two opposing pep-
tides toward Ang-(1-7) more effectively during a low sodium intake. Moreover,
blood pressure was the lowest in the group treated with ACE inhibitor and low-
salt intake. The findings from these studies corroborate well with previous con-
clusion that anti-hypertensive and cardio-renal protective effects of RAS block-
ade stemmed, at least in part, from Ang-(1-7) pathway activation [4648] and that
these effects may be more pronounced if followed by dietary sodium restriction
[49]. Further studies are clearly necessary to explore whether the beneficial effects
of dietary sodium alteration and/or pharmacological intervention indeed depend
on preferable ACE/ACE2 and ultimately Ang II/Ang-(1-7) balance in the target
organs.
10.3 ACE2: A Critical Enzyme Regulator in the Heart
The discovery of the biological effector peptide, Ang-(1-7) in 1988 represented the
first expansion of the classical RAS cascade in that it was the only other known
peptide member of the RAS to elicit some physiological function. However, the
formation of Ang-(1-7) remained elusive for several years. Welches and colleagues
[50] first showed that Ang-(1-7) could be formed from the traditional RAS precur-
sor peptide, Ang I, by endopeptidases including prolyl oligopeptidase (POP, E.C.
21.26), neprilysin (NEP, E.C. 24.11), and thimet oligopeptidase (TOP, E.C. 24.15).
While it was known that prolyl oligopeptidase could cleave the Pro7 -Phe8 bond of
Ang II, the studies were not supported by convincing in vivo evidence. Moreover,
studies by Yang et al. [51] found that prolyl carboxypeptidase (PCP, E.C. 16.2), a
lysosomal enzyme with an acidic pH optimum, could cleave Ang II into Ang-(1-7).
The hydrolysis appeared to be an intracellular cleavage, and the observation that the
acidic pH optimum of PCP of 5.0 provided some doubt as to the physiological role
for this enzyme in producing Ang-(1-7).
It was not until 2000, when two independent research groups described a
homolog of ACE, called angiotensin-converting enzyme 2 (ACE2) [52, 53], that
a viable Ang-(1-7)-forming enzyme from Ang II was discovered. Shortly after its
discovery, Vickers et al. [54] showed that ACE2 could cleave Ang II into Ang-(1-7)
with high affinity. ACE2 also cleaved apelin, des-Arg9 -bradykinin, and the opioid
peptide dynorphin A 1-13 with similar affinities, but its involvement in modulating
these peptides in vivo remains to be clarified. Subsequent studies revealed the gen-
eration of Ang-(1-7) in human failing heart tissue, which was dependent on Ang
II [55], suggesting that ACE2 was required for the cleavage of Ang II into Ang-
(1-7). Studies from our laboratory represented the first direct in vivo evidence for
ACE2s participation in hydrolyzing Ang II into Ang-(1-7) in hearts isolated from
both normal and hypertensive rats [10]. We further showed that the hypertrophied
hearts from hypertensive rats were almost completely reliant on ACE2 for the
production of Ang-(1-7) from Ang II, whereas ACE2 in the normal heart was of less
importance.
ACE2 is widely expressed in many tissues in humans [56] and rodents [56, 57],
including the heart. In addition, work from this laboratory first demonstrated that
cardiac expression of ACE2 mRNA was regulated by the actions of Ang II via an
AT1 receptor pathway [58]. A more recent study showed that the negative actions of
Ang II on cardiac ACE2 mRNA could be mimicked by the addition of endothelin-
1 and that both effects could be blocked by inhibitors of mitogen-activated protein
(MAP) kinase kinase 1, suggesting that Ang II or endothelin-1 activate ERK1/ERK2
to reduce ACE2 [59].
The importance of ACE2 in the regulation of cardiac function was determined
when Crackower and colleagues [29] demonstrated that genetic inactivation of
ACE2 in mice resulted in severe blood-pressure-independent systolic impairments
in cardiac function, which was associated with significant accumulation of circulat-
ing and cardiac Ang II. The concomitant genetic inactivation of ACE completely
rescued the ACE2-null cardiac phenotype, further implicating elevated Ang II in
cardiac dysfunction observed in the ACE2-null mice. These studies were the first
to support the in vivo importance of ACE2 in regulating cardiac function and Ang
II metabolism. In addition to these findings, two additional ACE2-null mice strains
were generated by separate groups [60, 61]. One strain exhibited cardiac dysfunc-
tion only in response to pressure overload, which was also associated with increased
cardiac Ang II [61]. The impairments in cardiac dysfunction were abrogated with
the co-administration of the AT1 receptor antagonist, candesartan. In contrast, Gur-
ley et al. [60] reported that genetic inactivation of ACE2 in 129/SvEv, C57BL/6, or
mixed mouse backgrounds did not induce any functional impairments in the heart,
suggesting that the importance of cardiac ACE2 may be dependent on the genetic
background of the animal model [62]. In this context, Mercure et al. [63] reported
that an eightfold increase in Ang(1-7) in the heart of transgenic animals was asso-
ciated with less ventricular hypertrophy and fibrosis than their nontransgenic litter-
mates in response to a hypertensive challenge.
A view from an opposite approach to determine the physiological importance
of ACE2 further favors a cardioprotective role for the enzyme. Indeed, the overex-
pression of ACE2 protects the heart from Ang II-induced cardiac hypertrophy and
myocardial fibrosis in rats [64]. Moreover, elevations in cardiac ACE2 exhibited a
partial rescue of the cardiac functional deficits induced by coronary artery ligation
in rats [65]. The cardioprotective persona given to ACE2 is further illustrated by
its regulation in pathological conditions. Three very important independent studies
showed that cardiac ACE2 was upregulated in both humans and rodent models of
heart failure [6668]. Moreover, we first showed that secreted ACE2 (sACE2) was
elevated in the cardiac effluent of hypertrophied hearts, suggesting that the enzyme
was attempting to protect the heart from progressing toward overt failure as is known
in the Ren-2 transgenic rats [10]. These data were very recently supported by human
studies that measured sACE2 activity in human plasma, and the authors showed that
the sACE2 was indeed markedly increased in patients diagnosed with heart failure
[69]. Intriguingly, Lambert et al. [70] recently demonstrated that the tumor necrosis
10 Ang-(1-7) and ACE2 127
factor convertase, ADAM17, participated in the shedding of ACE2 from the mem-
brane, and other studies have reported that ADAM17 is upregulated in heart fail-
ure [71].
Collectively, the data on the physiological importance of cardiac ACE2 are clear:
it exerts a cardioprotective role from the early stages of cardiac hypertrophy through
overt heart failure, although its efforts may be insufficient to overcome the pro-
gression of heart disease. However, further studies are required to determine the
importance of the discovery of an endogenous ACE2 inhibitor [72], as well as the
emerging data that ACE2 is a functional receptor for the SARS coronavirus [73], as
these may provide alternative therapeutic targets for the treatment of cardiovascular
disease.
10.4 Angiotensin-(1-12)
Over the years, questions have been raised regarding the capability of cardiac and
vascular tissue to synthesize Ang II [7478]. The heart remains a critical example.
Although a large body of evidence suggests the existence of local tissue RAS in the
regulation of cardiac function and remodeling, most studies revealed low levels of
gene expression for both cardiac renin and Aogen [79]. Neither the identification
of renin in cardiac mast cells [80] excludes an uptake mechanism from the blood
compartment nor does the finding of renin activation by binding of prorenin to the
prorenin/renin receptor [8183] can be construed as evidence for local production of
cellular renin. Likewise, Aogen gene expression in cardiac tissue has been reported
at very low expression levels while the question of how much of the Aogen mRNA
is due to its presence in the endothelial cells of intracoronary vessels and cardiac
fibroblast has not been answered.
Our current view of the RAS as a complex system entailing several levels
of regulation and processing is now further expanded with the identification of
proangiotensin-12 [angiotensin-(1-12), Ang-(1-12)] as an upstream propeptide to
Ang I [84]. These investigators first isolated this novel Aogen-derived peptide from
the rat small intestine. Consisting of 12 amino acids, this peptide was termed
proangiotensin-12 based on its possible role as an Ang II precursor. Ang-(1-12)
constricted aortic strips and, when infused intravenously, raised blood pressure in
rats. The vasoconstrictor responses to Ang-(1-12) were abolished by either cap-
topril or the AT1 blocker CV-11974. Current studies from this laboratory now
demonstrate the existence of Ang-(1-12) in both the heart and the kidneys of
spontaneously hypertensive rats (SHR), primarily restricted to cardiac myocytes
and renal tubular cells [85]. In addition, the cardiac content of Ang-(1-12) was
significantly augmented in the heart of SHR compared to Wistar-Kyoto (WKY)
controls [85]. Moreover, an insight into the processing of Ang-(1-12) into Ang
I, Ang II, and Ang-(1-7) was accomplished by studying the effects of exoge-
nously administered Ang-(1-12) in isolated hearts from both normotensive and
hypertensive rat strains [86]. In these studies, we showed processing of
Ang-(1-12) into Ang I, Ang II, and Ang-(1-7). Moreover, in the group of WKY
and SHR investigated in this study, the addition of a specific renin inhibitor to the
preparation in no manner altered the production of angiotensins from Ang-(1-12).
These data showed that Ang-(1-12) is processed into the active angiotensin peptides
by a non-renin mechanism. While further work will be required to ascertain the bio-
logical role of Ang-(1-12), these data expand on our knowledge of the mechanisms
by which the RAS regulates the expression of angiotensins in tissues [8790].
10.5 Conclusions
The discovery of the counter-balancing ACE2/Ang-(1-7)/mas arm of the RAS has
expanded knowledge of the intrinsic mechanisms by which the system regulates
homeostasis and tissue perfusion in both physiology and pathology [91]. Rapid
advances in this field now suggest alternate approaches to suppress the patholog-
ical actions of Ang II by enhancing the counter-regulatory actions of Ang-(1-7),
augmenting the activity of ACE2, or both. Moreover, the discovery of Ang-(1-12)
in multiple tissues including the heart may provide additional mechanistic insights
that could lead to the better treatment and management of hypertension and heart
failure.
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Chapter 11
Kinin Receptors and ACE Inhibitors:
An Interrelationship
Ervin G. Erds, Fulong Tan, and Randal A. Skidgel
Abstract The beneficial effects of angiotensin I-converting enzyme (ACE)

inhibitors are due in part to augmenting the actions of bradykinin (BK) and Lys-
BK on their receptors (R). They inhibit kinin inactivation and thereby stimulate
the release of mediators such as prostaglandins, nitric oxide (NO), and others. In
addition to inhibiting an enzyme, ACE inhibitors affect BK Rs as allosteric effec-
tors in cultured cells, such as human endothelial cells. ACE inhibitors can poten-
tiate BK and ACE-resistant BK analogs actions on B2 Rs. They elevate arachi-
donic acid and NO release as indirect allosteric enhancers acting on a heterodimer
formed by human ACE and B2 R. This has been shown by co-immunoprecipitation,
immunohistochemistry and fluorescence resonance energy transfer (FRET). After
carboxypeptidase N or M removes the C-terminal Arg of kinins, the resulting des-
Arg9 -BK and des-Arg10 -Lys1 -BK are inactive on B2 Rs, but are agonists of B1 Rs.
Activation of this R leads to prolonged release of NO, synthesized by iNOS. ACE
inhibitors are also agonists of the B1 R at a Zn-binding sequence of this heptahelical
G protein-coupled R. The site of activation is different from that of the orthosteric
peptide ligands; it is on the extracellular loop 2 at residues 195199. Thus, ACE
inhibitors act as direct allosteric agonists. B1 Rs are present mainly in endothelial
and other cells after an inflammatory process or induced by cytokines, which also
bring about iNOS expression. While constitutively expressed eNOS activation via
B2 R results in a short burst of NO, the longer lasting NO release initiated by peptide
or ACE inhibitor ligands of the B1R may help to alleviate some detrimental effects
in the failing heart.
When we received the invitation to contribute to this volume on angiotensin-

converting enzyme (ACE) and kinins, we quickly found that an all-encompassing,
comprehensive review would be impossible; the number of abstracts dealing with
this topic and its ramifications, published during the past 5 years, is 10,677. In a
E.G. Erds (B)

Departments of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA
e-mail: egerdos@uic.edu

136 E.G. Erds et al.
Gordon Conference some years ago, I (EGE) surmised that the relationship between
bradykinin (BK) and angiotensin (Ang) reminded me of an old, bad marriage, where
the association is sometimes antagonistic, other times supportive, but mostly indif-
ferent. However, with changing morals and ACE inhibitors, this association became
a mnage trois, then, when adding receptors, a mnage quatre or more.
Initially, researchers worked on the renin angiotensin and kallikrein-kinin sys-
tems happily oblivious of each other. This ended when we showed that kininase II
and ACE are one and the same protein, having the dual action of releasing a C-
terminal His-Leu dipeptide from Ang I and Phe-Arg from BK [14] (Fig. 11.1).
ACE was discovered as a factor in horse plasma by Skeggs and associates [5],
and we found kininase II in a kidney microsomal fraction [1] and isolated it from
human plasma when looking for another enzyme, carboxypeptidase N [3].
ACE
Inhibitors
Bradykinin (Kallidin)
(Lys) des-Arg-bradykinin
g y ((Kallidin))
ACE
BK (Lys)
CPN (plasma)
(17)
BK ACE
(Inactive) BK IInhibitors
hibit
(19)
(18)
CPM
B2 Receptor B1 Receptor
Induction:
Injury
Cytokines
Endotoxin
Fig. 11.1 B1 and B2R agonists and their regulation by ACE, carboxypeptidase N and carboxypep-
tidase M. ACE inactivates BK to BK (1-7), inactive on both B1 and B2Rs. The action of plasma
carboxypeptidase N or membrane carboxypeptidase M on BK is required to generate des-Arg-kinin
B1R agonists and inactivate BK or kallidin as B2R agonists. B2 Rs are constitutively expressed
whereas B1R expression is induced by injury, cytokines, or endotoxin. ACE inhibitors enhance
B1R signaling in two ways: (1) they block degradation of BK/kallidin by ACE and thereby enhance
substrate levels for carboxypeptidase M/carboxypeptidase N generation of B1R agonists. (2) They
can directly bind B1Rs and activate signaling
11 Kinin Receptors and ACE Inhibitors: An Interrelationship 137
Other common links between the two systems were observed later, and at least
some of the effects of the kallikrein-kinin system appear to dampen the hyperten-
sive effects of the renin angiotensin system by activating hypotensive responses. It
follows that when ACE inhibitors are employed, the primary beneficial effects will
result from blocking Ang II release and BK inactivation. However, many labora-
tory experiments [6, 7] and the treatment of tens of millions of patients with ACE
inhibitors have revealed numerous effects [812] that could not be explained by only
blocking the enzymatic hydrolysis of the two main peptide substrates. Some pub-
lications suggested that ACE inhibitors may act somehow independent of enzyme
inhibition, also directly on the receptors of BK [13, 14]. Later, it was reported that
ACE inhibitors transmit a signal via the intracellular tail of ACE to enhance some
kinase activities, which yield elevated COX 2 expression [15].
11.1 ACE Inhibitors and Kinin B1 Receptors
We found in experiments done first with bioassays of surviving tissues and in cul-
tured cells that ACE inhibitors affect the two kinin receptors B1 and B2 (B1R and
B2R) in different ways [1618]. The B1R is usually expressed after noxious stim-
uli, or by adding cytokines to cultured cells [19] or by transfection [20], but some
cells (bovine lung endothelial or human fibroblasts) express constitutive B1Rs. The
endogenous ligands of B1R are desArg9 -BK and desArg10 -Lys1 -BK (des-Arg10 -
kallidin). When plasma carboxypeptidase N or cell membrane carboxypeptidase M
cleave BK, they remove the C-terminal basic amino acid arginine and thereby lib-
erate the ligands of B1 [19, 21, 22] (Fig. 11.1). ACE inhibitors directly activated
B1Rs and released NO in our experiments, most consistently in cultured human
endothelial cells, via iNOS [18, 23]. The real-time NO liberation measured with a
porphyrinic electrode was in the same range as that of the B1 ligands, desArg10 Lys1 -
BK and desArg9 -BK, in concentrations ranging from 1 nM to 1 M assessed at an
embarrassing n = 284 times [23] (Brovkovych et al., to be published). Enalaprilat,
quinaprilat, captopril, or ramiprilat were active, while lisinopril was not at the same
concentrations. This is attributed to structural differences, for example, to the posi-
tively charged -NH2 group in lisinopril [2325]. Although des-Arg10 Lys1 -BK had
higher affinity for the B1R than desArg9 -BK, the mistaken notion that affinity is
equivalent to potency led to statements that des-Arg10 Lys1 -BK is much more potent
than desArg9 -BK [26]. We and others [2729] found that the two peptide agonists
are equally active on human B1Rs. Thus, even if their affinities to the human recep-
tor are different, the des-Arg nona- and octa-peptide derivatives have about the same
efficacy [27].
The human B1R is directly activated by ACE inhibitors, even in the absence
of ACE expression, to generate NO and other mediator release [2325]. A zinc-
binding motif (HEMGH) in the active site of ACE is required for its activity and is
an important site of attachment of ACE inhibitors [30]. The human B1R contains
in its second extracellular loop (residues 195199) a similar HEAWH sequence, a
canonical Zn2+ -binding pentamer, which represents the target for B1R activation
by ACE inhibitors [18, 25]. Since the orthosteric peptide agonists of B1Rs and the
allosteric ACE inhibitor agonists activate the receptor at different sites, the ACE
inhibitor activity is blocked by agents or mutations that do not affect des-Arg-kinin
activity [2325, 27]. For example, a synthetic undecapeptide (LLPHEAWHFAR;
residues 192202 of the human B1R), which includes this pentamer, blocked B1R
activation by enalaprilat but not by des-Arg-kinin [25, 27]. When the H195 was
mutated to Ala in the human receptor, the activation by peptide agonist was unaf-
fected, but that by enalaprilat was much reduced [25] (Tan et al., to be published).
The increased enzymatic NO synthesis via B1R activation may also con-
tribute to ACE inhibitors therapeutic effects [31] even after an MI [32]. We
found that ACE inhibitor/B1R-mediated NO inhibited protein kinase C (PKC)
(Fig. 11.2) [27], which could also have beneficial effects on the failing heart [33].
The two endogenous peptide agonists of the B1R are also equally active in stimu-
lating this response, and inhibition of PKC [27] is independent of blocking Ang II
generation or BK metabolism by ACE.
ACE inhibitors can enhance B1R activation by another mechanism as well,
by elevating its ligand concentrations. Since ACE is the major kinin inactiva-
tor, its inhibitors will raise intact kinin levels and consequently the substrate
Fig. 11.2 Stimulation of B1Rs by peptide agonist or ACE inhibitor inhibits PKC. Human lung
microvascular endothelial cells were pretreated with cytokines IL-1 (5 ng/ml) and IFN- (200
U/ml) to upregulate B1R and iNOS expression. Cells were then stimulated with either des-Arg10 -
kallidin (DAKD; 10 nM) or the ACE inhibitor enalaprilat (EPT; 10 nM) for 20 min or pretreated
with the B1R antagonist des-Arg10 -Leu9 -kallidin (DALKD; 100 nM). Both DAKD and enalaprilat
inhibited PKC; inhibition was abolished by a B1R antagonist. Ordinate: PKC relative activity.
Data are expressed as mean SE (n = 3; done in triplicate). Reproduced from Stanisavljevic
et al. [27] with permission from the American Society for Pharmacology and Experimental
Therapeutics
concentration for carboxypeptidases M and N to deliver more B1R agonists [22,

25, 34] (Fig. 11.1). In fact, without carboxypeptidase expression, B1R signaling
could not occur, indicating the B1R is a peptidase-activated receptor. Our recent
studies have shown carboxypeptidase M and B1Rs interact on the cell membrane
as determined by co-immunoprecipitation, crosslinking, and FRET analysis [20].
Furthermore, carboxypeptidase M and B1R are co-localized in lipid raft/caveolin-
enriched membrane fractions and disruption with methyl--cyclodextrin reduced
the B1R response. A monoclonal antibody to the C-terminal -sheet domain of car-
boxypeptidase M also reduced the B1R response to native kinins without inhibit-
ing carboxypeptidase M [20]. The intimate association of carboxypeptidase M and
B1R is thus required for efficient delivery of agonist from carboxypeptidase M to
the B1R. In bovine or human endothelial cells, B2R agonists stimulate a calcium
signal [20] or nitric oxide production [34] that are B1R- and carboxypeptidase
M-dependent. Based on the crystal structure of carboxypeptidase M and model-
ing [35], the glycosylphosphatidylinositol anchor and positively charged residues
on one face of carboxypeptidase M likely mediate its interactions with the mem-
brane phospholipids, resulting in the proper orientation of its C-terminal -sheet
domain in relation to the B1R to allow proper interaction and efficient delivery of
agonist. Although carboxypeptidase M is expressed constitutively in many cell types
[36, 37], its expression can be increased about two- to threefold in cytokine-treated
human endothelial cells [34], thus further enhancing B1R signaling in inflammatory
or pathological responses.
11.2 ACE Inhibitors and Kinin B2 Receptors

The B2R, compared with the B1R, is the more ubiquitous receptor of intact BK and
Lys-BK as it is constitutively expressed [19, 38]. Besides protecting BK from enzy-
matic breakdown by ACE (kininase II), ACE inhibitors can enhance BK actions by
potentiating the activation of B2R and by resensitizing the receptor desensitized by
the agonist BK [17, 39, 40] (Fig. 11.3). To differentiate the two effects, we tested
several ACE-resistant analogs [4042]. These either had a different covalent bond
between Pro7 -Phe8 of BK (Bkan) to make it resistant to ACE [43] or are peptides
we synthesized with an enlarged BK N-terminus either coupled to soluble dextran
[41] or dansylated at the - and -NH2 in Lys1 -BK (kallidin) [42]. The crystal struc-
ture of ACE revealed features that restrict the size of the peptide substrates it can
hydrolyze [44] while the B2R still reacts to the enlarged analogue ligands. When
Phe8 of BK was substituted with Tyr(Me), the resulting peptide was about 50%
resistant to human ACE [45]. These peptide agonists, especially BKan, which we
used frequently, could clearly separate the protection against enzymatic breakdown
from an indirect augmentation of BK activity at its B2R by acting on ACE [46].
With BK analogs, we found that ACE inhibitors are indirect allosteric enhancers
of B2R activity [47, 48], yielding increased mediator (e.g., NO) release; some ben-
eficial effects of ACE inhibitors on the failing heart can involve the activation
of both B1 and B2Rs [49]. When human ACE or human B2Rs were singly and
Active site lid

Conformational Change
N-domain ACE inhibitor
ACE
C-domain
B2 Receptor
Resensitization
Potentiation
Fig. 11.3 Schematic diagram illustrating the allosteric activation of B2Rs by ACE
inhibitors mediated by conformational change of ACE. ACE has two active-site domains
(N-or C-domain) attached by a bridge section, and the C-domain is followed by a stalk region, a
hydrophobic transmembrane helix, and a short cytosolic tail. Based on the crystal structure, ACE
has a deep active-site cleft largely closed to the exterior by a lid region (left) that would require
conformational change to allow access of substrates or inhibitor (right). Thus, binding of ACE
inhibitor to the N- or C-domain would alter the conformation of ACE, which is constitutively asso-
ciated with B2Rs in a heterodimer. This conformational change would then result in movement
of the B2R, likely mediated by the C-domain of ACE, resulting in enhanced mediator release or
resensitization of the receptor
separately transfected into cultured cells, which otherwise lacked the two proteins,
ACE inhibitors ceased to be allosteric enhancers. They worked as such only when
both enzyme and receptors were present on the same cells. The inhibitors, acting
through ACE, can increase B2R signaling, resulting in enhanced release of arachi-
donic acid or Ca2+ [40, 42, 50] (Fig. 11.3). Others have noticed that various agents,
including some so-called BK potentiating snake venom peptides [51], related to the
first clinically employed ACE inhibitor teprotide [52], can enhance BK effects on
isolated tissues, even if they do not block BK inactivation [53, 54].
We obtained convincing initial pieces of evidence by using ancient bioassay
techniques. BK contracts the isolated guinea pig ileum isotonically [17, 55]. When
we added an ACE inhibitor at the peak of the contraction into the tissue bath, within
seconds it doubled the magnitude of contraction (Fig. 11.4). This happened despite
the very low kininase activity of the guinea pig preparation (t 1/2 = 1216 min for
BK), thus it could not have been due to blocking BK inactivation. Using an isolated
guinea pig atrial preparation, enalaprilat augmented the positive inotropic effects of
BK, also independent of its inactivation [46].
Fig. 11.4 Resensitization of B2R causing contraction of guinea pig ileum by ACE inhibitor.
Bradykinin (BK; 50 nM) contracts an isolated segment of the guinea pig ileum. BK is immedi-
ately potentiated by the ACE inhibitor enalaprilat (EPT; 0.2 m) which, given at the height of
isotonic contraction, further enhanced it. Reproduced from Erds et al. [17] with permission from
Elsevier
BK as an agonist induces phosphorylation of Ser residues in B2Rs [56]. ACE

inhibitors diminish the phosphorylation [48], but not by acting directly on the B2R
in cultured cells. From these and other experiments with specific inhibitors, we con-
cluded that activation of B2R by BK [46, 48] and its potentiation or reactivation of
the desensitized B2R by ACE inhibitors initiate different signal transduction path-
ways [57]. In our experiments, othosteric agonists (BK) reduce receptor signal to
a second dose of agonist, which is tachyphylaxis or desensitation [57]. However,
added allosteric modifiers modulate this effect. Resensitization by ACE inhibitors
[58] restores sensitivity to B2R agonist BK. ACE inhibitor elevates the efficacy of
B2Rs, and allosteric interactions between ACE and the receptor stabilize it in a more
favorable conformation (Fig. 11.3).
To determine what regions of human 150180 kDa ACE participate in the poten-
tiation of BK effects [47], mutated and chimeric ACE were synthesized [40] and
N- and C-domain-specific reagents were used. ACE inhibitors potentiated BK by
acting through either the N- or C-domain. The active center of ACE was involved in
ACE effects because monoclonal antibodies [47] or sequestering agent binding the
Zn2+ cofactor also potentiated the peptide agonists. But adding enalaprilat (1 M)
to the treated and inhibited enzyme further augmented BK effects [46].
For ACE inhibitors to be allosteric enhancers of B2R, both ACE and B2R would
have to be expressed close enough on the plasma membrane to transmit allosteric
effects induced indirectly by ACE inhibitors via ACE to the B2R [48] (Fig. 11.3).
We approached the problem using several techniques [40, 42]. First, we investigated
whether ACE and B2R interact by co-immunoprecipitation with polyclonal antibod-
ies against human ACE. We tagged B2R at its C-terminal end with green fluorescent
protein (GFP), to use an antibody elicited to GFP. ACE and the B2R fused with GFP
were coprecipitated from lysed CHO cells, either with antibody to ACE or GFP as
shown in Western blot. Next, we employed immunohistochemistry of fixed cells
and located ACE with antibody to human ACE (stained red) and the B2R by green
fluorescence, owing to the fused GFP. The enzyme and the receptor colocalized as
indicated by the appearance of intense yellow coloring of the merged images [42].
2000
ACE-CFP
Fluorescent Intensity of ROI

B2R-YFP
1500
1000
500
0
0 20 40 60 80
Bleaching Time (s)
Fig. 11.5 FRET between ACE and B2 receptors. CHO cells co-expressing human ACE-CFP and
B2-YFP were analyzed, and linear unmixing of CFP and YFP emission spectra was done with
the Zeiss LSM-510 META detector. YFP fluorescence was photobleached in the region of interest
by scanning with a 514 nm laser, and post-bleach images were collected. The increase in donor
(ACE-CFP) intensity concomitant with the decrease in acceptor (B2R-YFP) fluorescence following
bleaching indicates energy transfer (FRET). This shows that ACE and B2Rs colocalize within
10 nm on the cell membrane
We then employed fluorescence resonance energy transfer (FRET) to gain an esti-

mate of the closeness of ACE and B2R on the membrane. For this we fused yellow
fluorescent protein (YFP) with B2R as acceptor and cyan fluorescent protein (CFP)
with ACE as donor. We measured FRET as enhanced fluorescence of the donor after
acceptor photobleaching. The significant increase in the ACE-CFP signal after pho-
tobleaching B2R-YFP (Fig. 11.5 ) indicates that the fluorophores on ACE and B2R
were within 10 nm on the cellular plasma membrane. Thus, besides the well-studied
phenomena that many receptors can form homo- or heterodimers or oligomers [59],
human ACE and B2R can also be considered a heterodimer. This complex formation
by ACE and B2 should be a bimolecular reaction, which depends on the concentra-
tion of the reactants. If ACE is present in excess, ACE inhibitors could acceler-
ate the activation of B2R by kinins more because the reaction would proceed as
a pseudo-first order one. It also follows that when cells express many more B2Rs
than ACE, ACE inhibitors would not as effectively potentiate BK as an agonist of
B2R. These experiments indicate that ACE inhibitors acting on ACE induce a con-
formational change in B2R and thus become indirect allosteric enhancers of B2R
agonists activity [42].
In the FRET assays above, ACE and B2Rs were tagged with CFP or YFP at
their C-termini. B2R is a heptahelical G protein-coupled receptor whereas ACE is
anchored to the plasma membrane by a short transmembrane sequence and a cytoso-
lic tail, as a continuation of its C-domain [30] (Fig. 11.1). The N-domain of ACE
is connected to the C-domain by a so-called bridge-section, susceptible to some
proteases [6062] which can liberate an intact N-domain. ACE is released from
the plasma membrane by trypsin or secretase cutting at a stalk section [30, 63].
The ratio of extracellular amino acid sequence in ACE compared to the cell-bound
transmembrane and cytosolic portions is about 24:1. The distribution for human
B2R is quite the opposite. Its free N-terminus and extracellular loops, compared to
the transmembrane plus cytosolic sequences, yield a 1:13 ratio of free extracel-
lular to bound/intracellular portions. Thus, to establish whether the N-terminal
fluorescent tags would be close enough in the heterodimer to generate FRET. To
express B2R with a fluorescent protein at the N-terminus presented additional tech-
nical difficulties as attempts to express a simple fusion protein were unsuccessful.
To overcome this difficulty, we (Skidgel, Tan, Chen, Erds, to be published) fused
the coding sequence for a signal peptide [20] to the N-terminus of the fluorescent
protein, which facilitated the expression of B2R on the membrane. Experiments
carried out as above yielded FRET between N-terminally tagged ACE and B2R,
confirming our results with the C-terminal tags. As a negative control, one protein
was tagged at the N-terminus and the other at the C-terminus, yielding only a low
level of FRET through the membrane.
The precise mechanism by which ACE inhibitors allosterically modify B2R con-
formation via ACE is still being explored, but our hypothesis is that ACE inhibitors
induce a conformational change in ACE, which is then transmitted to the B2R by
virtue of their close association on the membrane (Fig. 11.3). This is supported by
several lines of evidence. First, ACE inhibitors induce phosphorylation of ACEs C-
terminal tail and activate signal transduction pathways that increase the expression
of proteins such as COX-2 [15]. Second, the two active domains of ACE exhibit
negative cooperativity so that binding of an ACE inhibitor to one domain alters
the conformation of the second domain, making it no longer accessible to a sec-
ond molecule of inhibitor [64, 65]. Third, the crystal structure of either the C- or
N- domains of ACE revealed the presence of two subdomains surrounding a deep
active site cleft largely closed to the exterior by a lid that would require confor-
mational change to allow access of substrates or inhibitor [44, 66]. Fourth, normal
mode analysis of the ACE structure showed intrinsic flexibility around the active
site, and a hinge mechanism was proposed to explain opening of the subdomains to
allow substrate/inhibitor binding [66]. Taken together, these data support our finding
that ACE inhibitors act as indirect allosteric enhancers of B2R signaling by binding
to ACE, which causes a conformational change in ACE transmitted to the receptor
via the heterodimer, enhancing receptor function (Fig. 11.3).
11.3 Other Considerations

The two BK receptors have different agonists and antagonists, and B2 agonists acti-
vate eNOS while B1R ligands activate iNOS [23, 34, 67]. B1 and B2Rs also have a
rather complex interrelationship. While both B1R and B2R contribute to the mainte-
nance of normal blood pressure, in B2R knockout mice constitutive B1R expression
is upregulated [68]. Vice versa, B2Rs can compensate for a lack of B1Rs in mice,
with myocardial infarction [69].
Carboxypeptidase M, which generates B1R agonist peptides, is closely associ-

ated with B1Rs on plasma membranes, and in this complex, B1Rs responded to B2
agonists [20, 34]. ACE inhibitors can increase B1R agonist (Des-Arg9 -BK, Des-
Arg10 -Lys1 -BK) release by carboxypeptidase N or M [20, 22, 70]. Hypothetically
ACE inhibitor could decrease Ang 1-7 appearance by blocking Ang II liberation
by ACE. ACE 2 releases Ang 1-7 mainly from Ang II [71], which is also a sub-
strate of prolylcarboxypeptidase [72]. But the conversion of Ang I to Ang 1-7
by human neprilysin (neutral endopepeptidase 24.11) may be enhanced by ACE
inhibitors, which elevate Ang I levels [73]. Although neprilysin concentration is
low in plasma and in endothelial cells [74, 75], it occurs abundantly in other tis-
sues and cells, for example, in the renal proximal tubules [76], fibroblasts [77] and
leukocytes [78].
11.4 Similarities in the Development of Concepts: Kinins

and Angiotensin
In addition to ACE (kininase II) and its inhibitors linking the renin and kallikrein
systems, more interactions were found and some novel concepts introduced recently.
For example, how does blood-borne renin, an aspartic protease having only one
well-defined substrate in plasma, angiotensinogen, become active when it is needed
to restore blood pressure after a precipitous drop or when it is not really needed,
causing clinical hypertension? [79, 80] Human renin circulates as 92% inactive
prorenin [81] with little evidence for a significant activation in circulation and,
anyhow, human renin does not cleave its plasma substrate human angiotensinogen
efficiently at blood pH. The discovery of reninprorenin receptors and the concept
of dual Ang receptors answered unasked questions and shifted the paradigm, as
described elsewhere in this volume.
Our concepts dealing with the kallikrein-kinin system developed in some ways
comparable to the renin angiotensin system. A good part of the actions of ACE
inhibitors are attributed to protecting BK or Lys-BK against enzymatic breakdown.
This requires that a free kinin be present to act on its receptor, usually B2, leading
to enhanced mediator release, e.g., NO, prostaglandins, endothelium-derived hyper-
polarizing factor (EDHF) [82]. This process starts with the activation of the pro-
enzyme, prekallikrein, by an activator [83]. Then kallikrein cleaves plasma kinino-
gen to release a kinin, which is a substrate of kininases. This complex cascade,
instead of being the only process available to deliver a BK agonist, may be bypassed
or shunted. As shown in cultured cells [45] and in rats genetically depleted of
kininogen (kallikrein substrate) [84], kallikrein can directly activate the B2R even
in the absence of kinin release. This can be enhanced [85] by ACE inhibitors if
ACE is also expressed in the same cells. Thus, besides the major function of ACE
inhibitors to block the kininase activity, as allosteric enhancers they can augment
the activities of some proteases acting on BK B2R directly without the intermediate
release of a peptide [84, 86].
11.5 Epilogue
The beneficial effects of ACE inhibitors are due in part to augmenting BK and Lys1 -
BK effects on their receptor. They inhibit their inactivation, potentiate mediator
release after receptor activation, and resensitize the B2R desensitized by peptide
agonist. On the B2Rs, ACE inhibitors can also be considered as indirect allosteric
enhancers via ACE.
After their C-terminal Arg is cleaved by carboxypeptidase M or N, the resulting
desArg9 -BK and desArg10 -Lys1 -BK lose affinity to the B2R and become ligands of
the B1R (Fig. 11.1). ACE inhibitors can also directly activate the B1R to cause a
prolonged NO release via iNOS [2325, 27]. Such an activation of iNOS via B1R
[2325, 34] can aid the failing heart. Overexpression of iNOS in hearts of transgenic
mice decreased infarct size and reduced the detrimental effects of reperfusion [87].
Besides Ang I and BK, ACE also cleaves the metabolites of the two peptides further.
Ang 1-9 and Ang 1-7 also are endogenous allosteric enhancers of B2R activation
via ACE, at least in cultured cells [47, 50, 57]. Ang 1-9 is converted to Ang 1-7, but
very slowly, and then Ang 1-7 is also broken down by ACE to Ang 1-5. The reaction
rates for these substrates are much lower, the specificity constants are 1/4th1/10th
of that of Ang I. BK is rapidly inactivated by ACE, and the split product BK 1-7 is
cleaved further by ACE to BK 1-5. This pentapeptide is considered to be the final
BK metabolite in human blood [83, 88] with an intact N-terminal Arg1 -Pro2 bond, in
contrast to publications emphasizing an aminopeptidase (cleaving N-terminal Arg)
as a main BK inactivator in human blood [89].
Our conclusions are more self-evident than strikingly revolutionary. Receptors
are like humans; they usually do not work singly and have immediate consequences,
which are mainly local.
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Chapter 12
Kinins and Cardiovascular Disease
Oscar A. Carretero, Xiao-Ping Yang, and Nour-Eddine Rhaleb
Abstract Autocrine, endocrine, and neuroendocrine hormonal systems are

important factors that regulate cardiovascular and renal function. Alteration of the
balance among these systems may result in hypertension and target organ damage.
Changes in this balance could be due to (a) genetic factors and/or (b) environmental
factors. Endocrine and neuroendocrine vasopressor hormonal systems, such as the
renin angiotensin system, aldosterone, and catecholamines, play a well-established
and important role in the regulation of blood pressure and the pathogenesis of some
forms of hypertension and target organ damage. The role of vasodepressor autacoids
such as kinins is less well established. However, there is increasing evidence that
vasodepressor hormones not only play an important role in the regulation of blood
pressure and renal function but may also oppose remodeling of the cardiovascular
system. Here we will primarily review the role of kinins, which are oligopeptides
containing the sequence of bradykinin. They are generated from precursors known
as kininogens by enzymes such as glandular (tissue) and plasma kallikrein. Some
of the effects of kinins are mediated via autacoids such as eicosanoids, nitric oxide
(NO), endothelium-derived hyperpolarizing factor (EDHF), and/or tissue plasmino-
gen activator (tPA). Acting via these mediators, kinins play an important role in
the regulation of cardiovascular and renal function as well as some of the cardio-
vascular and renal effects of angiotensin-converting enzyme (ACE) and angiotensin
type 1 receptor antagonists (ARB). A study of Utah families revealed that a dom-
inant kallikrein gene expressed as high urinary kallikrein excretion was associated
with a decreased risk of essential hypertension. Also, a restriction fragment length
polymorphism (RFLP) that distinguishes the kallikrein gene family in one strain
of spontaneously hypertensive rats (SHR) from normotensive Brown Norway rats
has been identified; in recombinant inbred substrains derived from these SHR and
Brown Norway strains, the RFLP marking the kallikrein gene family of the SHR
cosegregated with an increase in blood pressure. However, humans, rats, and mice
with a deficiency of one component of the kallikrein-kinin system or chronic block-
O.A. Carretero (B)

Hypertension and Vascular Research Division, Department of Medicine and Heart and Vascular
Institute, Henry Ford Hospital, Detroit, MI, USA
e-mail: ocarret1@hfhs.org

152 O.A. Carretero et al.
ade of the kallikrein-kinin system do not have hypertension. In the kidney, kinins
participate in the regulation of papillary blood flow and water and sodium excre-
tion. B2 -KO mice appear to be more sensitive to the hypertensinogenic effect of
salt. Kinins participate in the acute antihypertensive effect of ACE inhibitors; how-
ever, in general, they are not involved in the chronic antihypertensive effects of ACE
inhibitors except for the acute phase of mineralocorticoid-salt-induced hyperten-
sion. Kinins acting via nitric oxide (NO) participate in the vascular protective effect
of ACE inhibitors during neointima formation. In myocardial infarction produced
by ischemia/reperfusion, kinins play an important role in the reduction of infarct
size induced by preconditioning or ACE inhibitors. In heart failure secondary to
infarction, the therapeutic effects of ACE inhibitors are partially mediated by kinins
via the release of NO. The therapeutic effect of ARB in heart failure is partly due
to activation of angiotensin type 2 receptors via kinins and NO. Thus kinins could
play an important role in the regulation of cardiovascular and renal function as well
as in many of the beneficial effects of ACE inhibitors and ARB.
12.1 Introduction
Both genetic and environmental factors acting via intermediary phenotypes par-
ticipate in the regulation of blood pressure, the etiology of hypertension, and the
development of target organ damage. Vasoactive systems are an important compo-
nent of these intermediary phenotypes. They can act as local hormones (intracrine,
autocrine, and paracrine) or as endocrine and neuroendocrine systems. We use
the term intracrine to indicate hormones which act within the cells that synthe-
size them, such as reactive oxygen species (O2 ) and products of proto-oncogenes.
The term autocrine is used to indicate hormones which act on the cell membrane
receptors where they are produced, such as growth factors. The term paracrine
denotes hormones which act near the site where they are produced, such as kinins,
eicosanoids, nitric oxide (NO), and endothelium-derived hyperpolarizing factor
(EDHF). Endocrine refers to hormones such as aldosterone which are released into
the extracellular fluid and act on distant target tissues, though they can also act in
an autocrine and paracrine faction. Finally, neuroendocrine hormones such as cate-
cholamines are released by neurons and act near to or distant from the site of release.
Blood pressure is the result of a balance between vasopressor and vasodepres-
sor systems. Alteration of this equilibrium may result in: (a) hypertension, (b) tar-
get organ damage, (c) effective antihypertensive treatment, or (d) hypotension and
shock. Changes in this balance could be due to (a) genetic factors such as mutations
in one of the genes of the vasoactive system and/or (b) environmental factors which
alter the activity of vasoactive systems. Endocrine and neuroendocrine vasopres-
sor systems, such as the renin angiotensin aldosterone system and catecholamines,
play a well-established and important role in the regulation of blood pressure, the
pathogenesis of some forms of hypertension, and target organ damage. The role of
vasodepressor systems is less well established; however, evidence suggests that they
play an important role in the regulation of blood flow, renal function, the pathogen-
esis of salt-induced hypertension and target organ damage, and the cardioprotective
12 Kinins and Cardiovascular Disease 153
effects of angiotensin-converting enzyme (ACE) inhibitors and angiotensin recep-

tor blockers (ARB) [15]. Vasodepressor hormones such as kinins, eicosanoids, NO,
and EDHF act as local hormonal systems, opposing the effects of vasopressor sys-
tems. Some vasodepressor systems such as atrial (ANF), brain (BNP), and C-type
(CNP) natriuretic peptides may act as both endocrine and local hormones. Here we
will review the kinin-generating system and the role of kinins in: (1) regulation of
local blood flow; (2) water and sodium excretion; (3) regulation of blood pressure
and pathogenesis of hypertension; and (4) the therapeutic effects of ACE inhibitors
and ARB.
12.2 The Kinin-Generating System

Kininogenases such as glandular and plasma kallikreins are enzymes that gen-
erate kinins by hydrolyzing substrates known as kininogens, which circulate at
high concentrations in plasma. Kinins are rapidly destroyed by a group of pep-
tidases known as kininases (Fig. 12.1). Plasma and glandular (tissue) kallikrein
are potent kininogenases and are both serine proteases. A single gene encodes for
plasma kallikrein, and there is a large family of glandular kallikrein genes; how-
ever, KLK1 is the only glandular kallikrein that generates kinins (hereafter referred
to as glandular kallikrein, or simply kallikrein). Plasma kallikrein, also known as
Fletcher factor, is expressed mainly in the liver; in plasma it is found in the zymo-
gen form (prekallikrein) and differs from glandular kallikrein in its biochemical,
Glandular Kallikrein
Kininogenases
Plasma Kallikrein (enzymes)
N-site C-site
X-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-X Kininogens
(substrates)
Bradykinin
Kinins
Lys-Bradykinin
NEP-24.15 Kininase I
3.4.11.2 Kininases
Kininase II (ACE; 4.4.15.1) (peptidases)
Aminopeptidase NEP-24.11
3.4.11.2
3.4.11.9
Fig. 12.1 Site of kininogen cleavage (solid arrows) by the main kininogenases (glandular and
plasma kallikrein). The broken arrows indicate sites of kinin cleavage by kininases (kininase I,
kininase II, neutral endopeptidases 24.11 and 24.15, and aminopeptidases). (Modified from [1])
immunological, and functional characteristics. It preferentially releases bradykinin

from high-molecular-weight kininogen (HMWK), also known as Fitzgerald fac-
tor. Together with HMWK and Hageman factor, plasma kallikrein is involved in
coagulation, fibrinolysis, and possibly activation of the complement system. The
plasma kallikrein-HMWK system, acting through the release of bradykinin, could
be involved in the local regulation of blood flow and in some of the effects of ACE
inhibitors. On the other hand, patients with congenital deficiency of plasma HMWK
(Fitzgerald trait) have normal amounts of kinins in their blood [6]. (For a review of
the plasma kallikrein-HMWK system, see [79])
Kallikrein belongs to a family of serine proteases with very high homology; the
genes encoding for these enzymes are tightly clustered and arranged in tandem on
the same chromosome. The number of family members varies widely among mam-
mals; it is estimated that the kallikrein family contains at least 3 genes in humans,
20 in the rat and 2330 in the mouse, many of them pseudogenes [10]. Despite
the highly homologous amino acid composition of the serine proteases encoded by
the kallikrein gene family, most are not kininogenases and act on entirely different
substrates. For example, tonin, a rat enzyme of the kallikrein family, hydrolyzes
angiotensinogen and generates angiotensin II; prostate-specific antigen, a human
enzyme of the kallikrein family, hydrolyzes semenogelin, a high-molecular-weight
seminal vesicle protein [11, 12]. We have isolated a new member of the kallikrein
family from the submandibular gland [13, 14]. This protease produces contraction
of isolated aortic rings and (like tonin) also generates angiotensin II, suggesting that
localized regions of variability are important in determining substrate specificity
and possibly function of all enzymes of the kallikrein family. (For a review of the
molecular biology of the glandular kallikrein-kininogen system, see [1517].)
True kallikrein or KLK1 is encoded by a single gene having five exons and four
introns. Other members of the kallikrein gene family have a similar exonic and
intronic structure, with the splice junctions completely conserved. The 5 and 3
flanking regions have a high homology among the various genes; however, gene
regulation and site of expression are different, suggesting that small variations in the
nucleotide sequence of the 5 region are important in the regulation of expression.
The kallikrein gene is expressed mainly in the submandibular gland, pancreas, and
kidney; however, using the polymerase chain reaction, we have demonstrated its
mRNA in vascular tissue, heart, and adrenal glands though in smaller amounts [18,
19]. Kallikrein and kallikrein-like enzymes have also been found in the arteries and
veins [20], heart [21], brain [22], pituitary gland [23, 24], pancreas [25], intestine
[26, 27], salivary and sweat glands [28], spleen [29], adrenal glands [30], blood cells
[19], and the exocrine secretions of these structures. Some of them are probably true
kallikrein, while others may be separate members of the kallikrein family.
There is immunoreactive kallikrein in plasma, primarily the inactive form, with
only a small portion being in the active form [3135]. In humans [36] and rabbits
[37], 50% or more of urinary kallikrein is the inactive or zymogen form, while
in rats most is in the active form [38]. Kallikrein can release kinins from low-
molecular-weight kininogen (LMWK) and HMWK. In humans, kallikrein releases
lys-bradykinin (kallidin), whereas in rodents it releases bradykinin [39, 40].
Homozygous tissue kallikrein-deficient mice exhibit renal hypercalciuria and

become hypocalcemic under low-calium (Ca) diet conditions as a result of defec-
tive tubular Ca reabsorption [41]. However, B2 / mice treated with a B1 receptor
antagonist did not exhibit any change in urinary Ca excretion and adapted normally
to the low-Ca diet conditions, indicating that tissue kallikrein may be a physio-
logic regualtor of renal tubular Ca transport via a non-kinin-mediated mechanism.
In humans, a loss-of-function polymorphism in exon 3 of the tissue kallikrein gene
results in the substitution of an active-site arginine at position 53 to a histidine
(R53H), with substantial loss of kallikrein activity (5060% lower than in normal)
[42]. In these individuals there was increased Ca reabsorption in thick ascending
limb under baseline conditions that counteracted a defect in distal tubule Ca reab-
sorption. However, more pharmacogenetic studies are needed to examine whether
abnormal Ca regulation by the kidney in R53H individuals is linked causally to the
kallikrein mutation [42].
Kininogens (kallikrein substrates) are the precursors of kinins. In plasma there
are two main forms, characterized as LMWK and HMWK [43, 44]. Both are potent
inhibitors of cysteine proteinases such as calpain and cathepsins H, L, and B [45
47]. In the rat there is a third kininogen known as t-kininogen, because it releases
kinins when incubated with trypsin but not with tissue or plasma kallikrein. It is one
of the main acute reactants of inflammation in the rat. All kininogens also inhibit
thiol proteases, such as cathepsin M, H, and calpains [4851]. HMWK is involved
in the early stages of surface-activated coagulation (intrinsic coagulation pathway)
[7, 9, 52].
Kininases are peptidases found in blood and other tissues, which hydrolyze
kinins and other peptidic hormones [53]. The best known is angiotensin-converting
enzyme (ACE) or kininase II, which converts angiotensin I to II and inactivates
kinin substance P and other peptides [53, 54]. Another important kininase is neutral
endopeptidase 24.11 (NEP-24.11), also known as enkephalinase, which not only
hydrolyzes kinins and enkephalins but also destroys ANF, BNP, and endothelin
[55, 56]. Research performed in our laboratory suggests that it may be an impor-
tant renal kininase, at least in the rat [57]. Other kininases include MEP-24.15,
aminopeptidases, and carboxypeptidases; however, it is not known whether they
play an important role in the degradation of kinins in vivo. After inhibition of
most of these enzymes in vivo, plasma concentrations of endogenous kinins do not
increase significantly and their half-life remains less than 20 s, suggesting that other
peptidases are also important in kinin metabolism [58].
Kinins are oligopeptides containing the sequence of bradykinin in their struc-
ture and act mainly as local hormones, since they circulate at very low concen-
trations (150 fmol/ml) and are rapidly hydrolyzed by kininases. In tissues such
as the kidney, heart, and aorta, kinin concentrations are higher (100350 fmol/g)
[59], suggesting that kinins act mainly as local hormones. Eicosanoids, NO, EDHF,
tPA, and cytokines mediate at least some of the effects of exogenously adminis-
tered kinins [6064] (Fig. 12.2). At least two subtypes of kinin receptors have been
well characterized using analogs of bradykinin, B1 and B2 [65, 66]. These recep-
tors have been cloned and belong to the family of seven transmembrane receptors
KININOGEN
KININOGENASE
KININS
B2 - receptor B1 - receptor
2
Eicosanoids EDHF NO T-PA GLUT-1 & -4
cAMP K++-channels cGMP Plasmin Glucosei
Depressor / Natriuretic/Antitrophic
Inflammation
Fibrinolysis / ROS Scavenger/ O2 consumption
Pain/Fibrosis ?
Improve cardiac metabolism
Fig. 12.2 Kinins act via the B2 and B1 receptors. Most of the known effects of kinins are medi-
ated by the B2 receptor, which in turn acts by stimulating the release of various intermediaries:
eicosanoids, endothelial derive hyperpolarizing factor (EDRF), nitric oxide (NO), tissue plasmino-
gen activator (tPA), glucose transporter (GLU- 1 & -2 (modified from [234]))
linked to G-proteins [67]. B1 receptors are not present or only present at very low
density in normal tissues, but are expressed and synthesized de novo during tissue
injury, inflammation, and administration of lipopolysaccharides such as endotoxin.
In some species, including rabbits, they mediate contraction of the isolated aorta and
relaxation of mesenteric arteries. The main agonists for this receptor are des-Arg9 -
bradykinin and des-Arg10 -kallidin. B2 receptors mediate most of the effects of
bradykinin and are the main receptors for the agonists, bradykinin, and kallidin (lys-
bradykinin). Studies using kinin analogs with agonistic and antagonistic properties
in various tissues suggest the existence of other subtypes of receptors [6872] dis-
covered that substitution of D-phenylalanine for proline at position 7 of bradykinin
converts it into a specific antagonist for B2 receptors; while the substitution of Phe8
in des-Arg9 -BK by a residue with aliphatic (Ala, Ile, Leu, D-Leu, norleucine) or
saturated cyclic hydrocarbon chain (cyclohexyalanine) produced antagonists for B1
receptors [73, 74]. Further modifications have resulted in a very potent B2 recep-
tor antagonist with long-lasting effects in vivo, DArg0 -[Hyp3 -Thi5 -DThi7 -Oic8 ]-
bradykinin or icatibant (Hoe-140) [75], which has become an important tool for
studying the role of kinins. More recently, oral active kinin antagonists have been
developed for the possible treatment of inflammation, hyperalgesia, and perhaps
cancer [7680].
In humans, it has been reported that the B2 receptor is activated by kallikreins
and other serine proteases and that this effect is blocked by the kinin antagonist
icatibant [81]. Some ACE inhibitors potentiate the effect of bradykinin not only by
inhibiting its hydrolysis but also by cross-talk between ACE and the B2 receptor
[82]. The B2 receptor also forms heterodimers with the angiotensin type 1 receptor
(AT1 ), causing increased activation of the angiotensin receptor [83]. The stability of
this heterodimer is not affected by bradykinin, angiotensin, or their respective recep-
tor antagonists [84]. The B2 receptor also forms a complex with eNOS, inhibiting
the generation of NO, and this effect is reversed by bradykinin [85]. The pathophys-
iological role of these interactions of the B2 receptor is not known; however, it has
been reported that in preeclampsia there is an increase in AT1 and B2 heterodimers
that could mediate the enhanced response to angiotensin II [86]. Thus, in some sit-
uations these interactions of the B2 receptor could play a physiopathological role.
12.3 The Kallikrein-Kinin System in the Vasculature and in the

Regulation of Local Blood Flow
Arteries and veins contain a kallikrein-like enzyme, and both vascular tissue and
smooth muscle cells in culture contain mRNA for kallikrein [18, 20]. Vascular
smooth muscle cells in culture release both kallikrein and kininogen [87]. Thus
the components of the kallikrein-kinin system are present in vascular tissue, where
they could play an important role in the regulation of vascular resistance. Recently,
in isolated arteries of mice with deletion of the gene expressing kallikrein, blood
flow-induced dilatation was found to be significantly reduced compared to controls,
suggesting that the kallikrein-kinin system in the arterial wall participates in this
process [88, 89]. Also, in humans, a partial genetic deficiency in tissuel kallikrein
(R53H) was reported associated with an inward remodeling of the brachial artery,
which is not adapted to a chronic increase in wall shear stress. This form of arterial
dysfunction affects 57% of white population [90]. The effect of ACE inhibitors
on local potentiation of kinins vasodilator effect appears to be partly attributable
to their prevention of bradykinin degradation and subsequent increases in the pro-
duction of endothelium-derived relaxing factors (EDRFs) such as NO. In SHR and
in the canine coronary artery, ACE inhibitors potentiate the endothelium-dependent
relaxation evoked by bradykinin [91, 92]. This vasorelaxation appears to be associ-
ated primarily with increased release of NO.
When the arterial endothelium is removed, the smooth muscle cells begin to pro-
liferate in the media; they then migrate across the internal elastic lamina into the
intima, where they cause neointimal hyperplasia, mimicking some of the vascular
changes that occur in atherosclerosis. ACE inhibitors have been shown to inhibit
neointima formation [93, 94]. Blocking kinins or inhibiting NO synthesis lessens
the protective effect of the ACE inhibitor, suggesting that it may be mediated by a
local increase in kinins which stimulates the release of NO [95, 96].
Kinins play an important role in the local regulation of blood flow in organs rich
in kallikrein, such as the submandibular gland, uteroplacental complex, and kidney
[97100]. In rats nephrectomized 48 h earlier to exclude the renal renin angiotensin
system, use of an angiotensin I-converting enzyme (kininase II) inhibitor sig-
nificantly increased blood flow in the submandibular gland but did not affect
blood pressure. In contrast, 10 min after sympathetic stimulation of the gland

to increase kallikrein secretion in the vascular compartment, the ACE inhibitor
markedly decreased blood pressure and increased kinin concentrations in arterial
blood [35, 101]. Changes in both blood flow and blood pressure were blocked by
antibodies to kinins and kallikrein. The effect of the ACE inhibitor on basal glandu-
lar blood flow was also blocked by a kinin antagonist [97]. At low doses the antag-
onist caused no significant change in blood flow when the ACE inhibitor was not
administered, whereas at high doses basal blood flow decreased significantly. These
data suggest that in organs rich in kallikrein, kinins play a role in the regulation
of basal blood flow. Studies using kinin antibodies and antagonists clearly indicate
that kinins act as paracrine hormones, regulating blood flow within the gland. These
studies also indicate that the effect of ACE inhibitors on blood flow is mediated by
kinins [97]. In nephrectomized pregnant rabbits infused with an angiotensin antag-
onist to block the uterine renin angiotensin system, ACE inhibitors increased both
uterine and placental blood flow and immunoreactive PGE2 , whereas these effects
were blocked by a kinin antibody [98]. This suggests that endogenously generated
kinins play a role in the regulation of uterine blood flow, either directly or through
the release of prostaglandins.
In conclusion, in organs rich in glandular kallikrein, such as the submandibular
gland and uteroplacental complex, kinins appear to play an important role in the
regulation of blood flow, especially when ACE is inhibited. In addition, the local
arterial kallikrein-kinin system also participates in the regulation of blood flow and
in the vascular protective effect of ACE inhibitors.
12.4 Kinins in the Regulation of Renal Blood Flow
Kinins may also play an important role in the regulation of renal blood flow.
Blocking renal kinins by infusing low doses of a kinin antagonist into the renal
artery of sodium-depleted dogs decreased renal blood flow and autoregulation of
the glomerular filtration rate (GFR) without changing blood pressure [102]. The
changes in renal blood flow were blocked by prior inhibition of ACE, suggesting
that either those changes were mediated by renin release due to an agonistic effect
of the kinin antagonist or renal kinins may have increased when ACE was inhib-
ited, thereby competing more effectively with the antagonist. The changes in GFR
autoregulation were not altered by the ACE inhibitor and may have been due to
a change in either the relationship between afferent and efferent glomerular arte-
riolar resistance or the coefficient of filtration [102]. We have recently shown that
the vasodilator effect of bradykinin in the renal efferent arteriole is mediated by
cytochrome P450 metabolites of arachidonic acid called epoxyeicosatrienoic acids
(EETs) and that bradykinin stimulates the glomeruli to release another cytochrome
P450 metabolite, the vasoconstrictor eicosanoid 20-hydroxyeicosatetraenoic acid
(20-HETE), and also an unidentified vasodilator prostaglandin, which together par-
ticipate in regulation of the downstream glomerular circulation and perhaps in the
regulation of GFR [103, 104].
We examined the role of kinins in the regulation of renal blood flow distribution
using a laser-Doppler flowmeter [99]. The kinin antagonist lowered papillary blood
flow without altering outer cortical blood flow, suggesting that intrarenally formed
kinins are important in regulating blood flow in the inner medulla. This study also
showed that renin angiotensin system (RAS) plays an important role in the regula-
tion of papillary blood flow, since after kinins were blocked, enalaprilat increased
flow significantly. We also found that when we inhibited both ACE and neutral
endopeptidase-24.11 (NEP-24.11), papillary blood flow increased by 50%, com-
pared to 25% when they were inhibited separately. These increases were blocked
by the kinin antagonist, indicating that the augmented papillary blood flow induced
by both ACE and NEP-24.11 inhibitors is mediated by increased kinin concentra-
tions in the interstitial space. We observed no consistent effect on water or sodium
excretion; however, water excretion tends to decrease in animals treated with a kinin
antagonist.
In anesthetized rats, blocking kinins decreased renal blood flow [100]. In dogs,
when kallikrein excretion was stimulated by sodium deprivation, a kinin antago-
nist (given intrarenally) partially blocked the effect of enalaprilat on renal blood
flow [105]. This suggests that although blockade of the renin angiotensin system
accounted for a significant portion of the increase in renal blood flow caused by the
ACE inhibitor, a substantial component was contributed by endogenous kinins. Sim-
ilar results were reported in rats in which the kallikrein-kinin system was stimulated
by deoxycorticosterone [106].
In the kidney, kinins play a minor role in the regulation of blood flow; however,
when the kallikrein-kinin system is stimulated by low sodium intake or mineralo-
corticoids, or when endogenous kinin degradation is inhibited, kinins appear to par-
ticipate in the regulation of renal blood flow [107, 108]. In addition, the data suggest
that kinins play an important role in the regulation of papillary blood flow, and that
during reduction of renal perfusion pressure, kinins may aid in the regulation of the
GFR [109].
12.5 Kinins in the Regulation of Water and Electrolyte Excretion

Renal kallikrein is located in the connecting cells of the connecting tubule; it is
released in significant amounts in this segment of the nephron and excreted in the
urine (Fig. 12.3) [37, 110, 111]. Kallikrein releases kinins into the lumen of the dis-
tal nephron, either from filtered kininogen or kininogen produced in the principal
cells of the distal nephron [112, 113]. Kinin receptors are also present in the col-
lecting duct [114]. In addition, kallikrein is released on the basolateral side of the
nephron, where it may liberate kinins from plasma kininogens [115]. The interstitial
renal fluid contains a high concentration of kinins [116]. The role of kinins in the
regulation of water and sodium excretion has been studied by increasing intrarenal
kinins, blocking kinins, or deleting the genes expressing kinin receptors or tissue
kallikrein [41, 117120]. Infusion of kinins into the late proximal nephron doubled
excretion of simultaneously administered 22 Na [117], and that part of this effect was
Fig. 12.3 Localization of the kallikrein-kinin system, renin, and prostaglandin in the nephron
(right brackets), anatomical subdivisions or the nephron (outer left brackets), and type of cells
found in the distal nephron (inner left brackets). PGE2 = prostaglandin E2 ; PGI2 = prostacyclin
mediated by prostaglandins [121], while infusion of a kinin antagonist into the late
proximal nephron reduced 22 Na recovery significantly [122]. After systemic admin-
istration of phosphoramidon, an inhibitor of NEP-24.11 (a major kininase in the
nephron), urinary excretion of kinins doubled; diuresis increased by 15% and natri-
uresis by 37% [57]. Although these data support the hypothesis that increased kinins
in the nephron participate in intrarenal control of water and electrolyte excretion, it
is also possible that the effect of this peptidase inhibitor is mediated by blocking
hydrolysis of other peptides such as atrial natriuretic factor (ANF) [123].
Infusion of aprotinin inhibited the enzymatic activity of urinary kallikrein but
did not affect acute water or electrolyte excretion in euvolemic and sodium- or
water-expanded rats (124]. A transient decrease in sodium excretion has been
observed during aprotinin administration in mineralocorticoid-treated rats [125].
Infusion of kinin antibodies into saline-expanded rats decreased sodium excretion
[118]; however, caution should be used in interpreting this finding, since antibod-
ies may stimulate release of histamine, cause an anaphylactoid reaction, or form
a high-molecular-weight complex with kininogen, which is then deposited in the
nephron, any of which might alter water and sodium excretion. To avoid these
problems, we use Fab fragments of kinin antibodies, which are rapidly distributed
in the extracellular fluid and excreted by the kidney; moreover, they do not form
high-molecular-weight complexes or activate complement and other proteolytic

systems in plasma, thus reducing the risk of anaphylactoid reactions. In unanes-
thetized rats, the Fab fragments blocked 70% of the effect of an injection of 100 ng
bradykinin on blood pressure and appeared rapidly in the urine, suggesting that they
block the effect of kinins not only in the vascular and interstitial spaces but also
in the lumen of the distal nephron. Using these Fab fragments and a kinin antag-
onist, we studied a model in which the renal kallikrein-kinin system is stimulated,
namely, DOCA-salt-treated rats. Both the Fab fragments and kinin antagonist signif-
icantly decreased urine volume and increased urinary osmolarity; however, only the
Fab fragments significantly lessened urinary sodium excretion, but without affect-
ing blood pressure, renal blood flow, or GFR [108]. The antidiuretic effect of the
Fab fragments and kinin antagonist may be due to blockade of kinins in the vascular
interstitial space of the kidney, since the antagonist is likely hydrolyzed in the prox-
imal tubule and does not reach the lumen of the distal nephron. On the other hand,
the antinatriuretic effect of Fab fragments of kinin antibodies on sodium excretion
may be due to blockade of kinins in both the vascular/interstitial and the urinary
compartments, and only the latter compartment, since the antibody appeared in the
urine and the antidiuretic effect was not observed with the antagonist. Thus kinins
may aid in the regulation of water and sodium excretion when the kallikrein-kinin
system is stimulated. In normal non-anesthetized rats, inhibition of kinin release in
the lumen of the nephron by Fab fragments of monoclonal antibodies to kallikrein
causes urinary PGE2 , UV, and UNa V to decrease. The changes in UV and UNa V
mimic those of PGE2 , suggesting that the natriuretic and diuretic effects of kinins
are mediated in part by PGE2 [119].
Tissue kallikrein-deficient mice were reported to lack the 70-kDa form of -
ENaC (epithelial sodium channel) consistent with reduced renal ENaC activity in
tissues that normally express tissue kallikrein, such as in cortical collecting duct.
However, in mice lacking B2 receptors, the abundance of the 70-kDa form of
-ENaC was increased, indicating that its absence in tissue kallikreuin-deficient
mice is not kinin-mediated [126]. In vitro, stimulation of the release of EDRF
from endothelial cells by bradykinin or acetylcholine increases cGMP content and
inhibits Na+ transport by cortical collecting duct cells [127]. In vivo, stimulation of
EDRF release by bradykinin induces natriuresis and diuresis without affecting the
GFR [128]. In conclusion, kinins acting as local hormones play a role in the regula-
tion of renal hemodynamic and excretory function, either directly or via the release
of PGE2 and EDRF.
12.6 Kinins as Regulators of Blood Pressure and Pathogenesis of

Hypertension
The development of antibodies to kinins and kallikrein, kinin antagonists, and kin-
inase inhibitors, as well as gene knockout (KO) models of the kallikrein-kinin
system and the discovery of kininogen-deficient rats and humans with various
spontaneous mutations of the system, has allowed us to study the role of kinins
in various physiological and pathological conditions. The role of the kallikrein-

kinin system in the pathogenesis of hypertension has been studied by (1) mea-
surements of the various components of the system, (2) the use of bradykinin
B2 receptor antagonists, (3) using mice in which the B1 , B2 , or both B1 and B2
receptors have been deleted by homologous recombination, (4) deletion of the tis-
sue kallikrein gene, and (5) using rats deficient in kininogen. Decreased activ-
ity of the kallikrein-kinin system may play a role in hypertension. Low urinary
kallikrein excretion in children is one of the major genetic markers associated
with a family history of essential hypertension, and children with high urinary
kallikrein excretion have less probability of a genetic background of hyperten-
sion [129132]. Also, a restriction fragment length polymorphism for the kallikrein
gene family in spontaneously hypertensive rats has been linked to high blood
pressure [133], and urinary kallikrein excretion is decreased in several models of
genetic hypertension. Urinary and/or arterial tissue kallikrein are also decreased in
renovascular hypertension and genetically hypertensive rats [134137]. Although
these reductions may be secondary to increases in blood pressure, decreased uri-
nary kallikrein in normotensive children of patients with essential hypertension
and in genetically hypertensive and Dahl salt-sensitive rats prior to the develop-
ment of hypertension [138142] suggest that the cause of these decreases may be
different.
Kinins circulate in concentrations of approximately 550 pg/ml of blood [6].
These concentrations need to be increased to at least 100 pg in humans [143] and
1,000 pg in rats [144] to cause acute decreases in blood pressure. Although blood
kinin concentrations may increase in some physiological and pathological situa-
tions, they seldom reach levels that could explain changes in blood pressure, save for
exceptional experimental conditions such as stimulation of the sympathetic nerve of
the submandibular gland in animals treated with ACE inhibitors (see section on
blood flow regulation). Thus kinins would have to act as paracrine hormones, regu-
lating local vascular resistance and organ function.
In early studies, acute administration of a kinin antagonist at high doses increased
blood pressure in most rats tested, while a vasodepressor effect was observed in
some [145]. Using a more potent antagonist [146], also at high doses, we found that
it produced a transient biphasic response: first a small pressor effect, followed by a
depressor effect [147]. At smaller doses, though still sufficient to block exogenous
bradykinin, the same antagonists did not alter normal blood pressure. These stud-
ies appear to be somewhat compatible with the hypothesis that kinins play a role
in the regulation of blood pressure. However, in order to demonstrate the pressor
effect, the kinin antagonist has to be used at much higher doses than those needed to
block the vasodepressor effect of exogenous bradykinin. High doses may be needed
to displace kinins bound to tissue receptors. We must be cautious in interpreting
these data, since we cannot rule out the possibility that these kinin antagonists have
a vasopressor effect, which is unrelated to kinin-blocking activity. Studies by our
group using kinin antibodies or their Fab fragments showed that although they par-
tially block the vasodepressor effect of kinins, they do not cause acute changes in
blood pressure.
Normal blood pressure and cardiovascular are often observed in HMWK-

deficient rats, B1 / or B2 / mice [148150]. However, in mice lacking tissue
kallikrein, despite having normal blood pressure, experience very obvious abnormal
structure and function of the heart [89]. Chronic blockade of B2 kinin receptors with
a potent and selective B2 antagonist, icatibant, did not increase blood pressure under
normal conditions or under conditions that favor the development of hypertension
in rats, such as (1) chronic infusion of a subpressor or pressor dose of Ang II, (2) a
high-salt diet, (3) or mineralocorticoids and salt [149, 151]. However, these results
are not universal [152155].
Mice with the bradykinin B2 receptor deleted by homologous recombination
(gene knockout) have normal blood pressure (Fig. 12.4). However, they develop
hypertension when fed a high-sodium diet (8%) for at least 2 months [156, 157].
Thus low kinin activity may be involved in the development and maintenance of salt-
sensitive high blood pressure. However, in these mice hypertension induced by min-
eralocorticoids (renin independent) or aortic coarctation (renin independent) was not
exacerbated [158]. Also, it has been reported that as these mice grow older, they also
develop hypertension and left ventricular hypertrophy even on a normal sodium diet
[159]. However, we were unable to confirm that ablation of B2 kinin receptors ren-
ders mice spontaneously hypertensive [148]. Others were also unable to confirm the
hypothesis that B2 kinin receptors are a major component in the maintenance of
normal blood pressure and cardiac structure [120, 157, 158, 160]. Mice deficient in
kinin B1 , B2, or B1 /B2 receptors or tissue kallikrein had similar blood pressure to
wild-type controls, confirming that kinins are not an important determinant of blood
pressure [160]. Moreover, mice lacking both B1 and B2 receptors did not present
140
Mean Blood Pressure (mm Hg)
130 B2 +/+
B2 /
120
110
100
Night Day Night Day Night Day
90
Fig. 12.4 Diurnal and nocturnal mean blood pressure of bradykinin B2 receptor knockout (/)
and wild type (+/+) mice. Blood pressure was measured 24 h by a telemetric system (Rhaleb N-E
and Carretero O.A. unpublished data)
any significant change in blood pressure after an increase in dietary salt intake [161].
The latter contrasts with previous observations, demonstrating a chronic hyperten-
sive effect of high-salt diet in B2 / mice [156, 157]. High-salt diet duration could
play an important factor in these differences. Indeed, Alfie et al. [156] could not
observe hypertension until after 8 weeks of high-salt diet, and Cervenka et al. [157]
started high-salt diet during gestation period and continued for up to 4 months of
age. On the other hand, B1 B2 / were subjected to high salt for only 5 weeks
[161]. It is unknown whether B1 / develop hypertension when given high salt.
Furthermore, wild-type mice or mice in which the gene expressing tissue kallikrein
has been deleted exhibited similar increase in blood pressure when submitted to
renovascular hypertension [162], further supporting the minor role of kallikrein-
kinin system in the control of blood pressure under basal conditions or during
hypertension.
In kininogen-deficient Brown Norway Katholiek rats (BNK), administration of
mineralocorticoids and salt or angiotensin II reportedly causes blood pressure to
increase similarly to rats with a normal kallikrein-kinin system [149]. This contra-
dicts with other reports [152154].
In conclusion, these studies suggest that kinins do not play an important role
in the regulation of normal blood pressure or in the pathogenesis of hyperten-
sion, although they may be involved in the pathogenesis of salt-induced hyper-
tension. Overall, chronic blockade of the kallikrein-kinin-system does not appear
to cause hypertension or potentiate hypertensinogenic stimuli, though the data are
inconsistent.
12.7 Role of Kinins in the Antihypertensive Effect of ACE

Inhibitors
Inhibition of kinin and degradation of other oligopeptides may contribute to the anti-
hypertensive effect of ACE inhibitors. While blockade of angiotensin II formation
appears to be important in this regard, the role of kinins is less well established.
Orally active ACE inhibitors are effective antihypertensive agents, not only in high-
renin hypertension but also in clinical and experimental models in which the renin
angiotensin system has not been pathogenetically implicated [163, 164]. Thus some
effects of ACE inhibitors may be mediated by a local renin angiotensin system,
kinins, or some other undetermined mechanism, since ACE can hydrolyze other
peptides (Fig. 12.5). ACE inhibitors may also potentiate the effect of kinins by a
direct interaction with the kinin B2 receptor [82].
Blood kinins are unchanged or moderately increased after the administration of
ACE inhibitors [1, 165, 166] (for review, see [167, 168]). Kinins in the urine report-
edly increase more consistently after the administration of ACE inhibitors, indicat-
ing that their concentration in renal tissue likewise increases [57, 169172]. This
in turn may contribute to the antihypertensive effect of ACE inhibitors by altering
renovascular resistance and increasing sodium and water excretion.
HYPOTHETICAL MECHANISMS OF ACTION OF

ANGIOTENSIN CONVERTING ENZYME INHIBITORS
ACE
INHIBITORS
Other Direct
Ang-II Kinins Ac-SDKP
Substrate Effect
Direct Direct? Ang-I / Ang 1-7

Direct
PGI2 Inh fibrob. Opioid Pept. Bk receptor
Aldosterone
NO proliferat. Substance P Scavenger O2
Sympat. Transm.
EDHF Inh collagen Neurotensin NO
TxA2 /PGH2
Glucose upt. deposition Chemot. Pept. Conductance
Growth Factors
tPA LHRH, etc
O. A. Carretero, HFH, Detroit, MI
Fig. 12.5 ACE has multiple substrates, and inhibition of their hydrolysis may explain the cardio-
protective effect of ACE inhibitors
Many studies have assessed the role of kinins in the acute antihypertensive effect
of ACE inhibitors. In various experimental models of hypertension, the acute antihy-
pertensive effect of ACE inhibitors is attenuated by blocking kinins with either high
titers of kinin antibodies [173175, 174, 176] or with a B2 kinin receptor antagonist
[165, 166, 177]. Kinin antagonists also partially reverse the antihypertensive effect
of ACE inhibition in rats with renovascular hypertension [176]. However, deficiency
in B2 kinin receptors did not affect the antihypertensive effect of ACE inhibitors
in mice with renovascular (2 kidney-1 clip) hypertension (Fig. 12.6). This is not
surprising since it is well established that renin angiotensin system plays a major
role in the development of renovascular hypertension. We assessed the influence of
kinins on the acute antihypertensive effect of enalaprilat in rats with severe hyper-
tension induced by aortic ligation between the renal arteries [166]. In this model,
renin plays an important role in the pathogenesis of hypertension [163]; however,
acute and severe hypertension can produce endothelial damage that may lead to acti-
vation of plasma kallikrein and increased kinin formation. We found that enalaprilat
lowered mean blood pressure by 48 6 mm Hg in the controls and 21 4 mm Hg
in the kinin antagonist group, which is a significant difference (p < 0.01); however,
kinin concentrations in arterial plasma were not significantly altered by the ACE
inhibitor (41 10 vs. 68 20 pg/ml) (Fig. 12.7). As indicated earlier, if mean
blood pressure in the unanesthetized rat is to be decreased, kinins in arterial blood
must reach at least 1,000 pg/ml [144]. Thus the effect of the ACE inhibitor may be
due to an increase in tissue kinins, which could act as a paracrine hormonal system
regulating vascular resistance. Cachofeiro et al. [165] demonstrated that pretreat-
ment with either a bradykinin antagonist or a NO synthesis inhibitor attenuated the
acute antihypertensive effect of both captopril and ramipril in SHR, but pretreatment
Fig. 12.6 Antihypertensive Sham (n = 5)

effect of ACE inhibitor in 2K-1C (n = 4) *
2K-1C + ramipril (n = 5)
*
bradykinin B2 receptor
knockout (/) mice. Mice 150 Surgery
Systolic Blood Pressure (mm Hg)

with 2 K-1C hypertension *
were given plain water 140 * *
* *
(vehicle) or water mixed with *
ACE inhibitor, ramipril
130
(4 mg/kg/day) to drink 5
weeks after blood pressure
was increased. ACE inhibitor 120 *
normalized blood pressure in
B2 / hypertensive mice. 110
p < 0.001, 2 K-1C versus **
sham; p < 0.001, 2 K-1C + 100 **
ramipril versus untreated ** **
2 K-1C (Rhaleb N-E and Vehicle or ACEi
90
Carretero O.A. unpublished
data)
al 1 2 3 4 5 6 7 8 9
Bas
Duration (weeks)
Fig. 12.7 Role of kinins in

160
Blood Kinin Concentration
the acute antihypertensive

effects of an ACE inhibitor 140
(enalaprilat) in rats with 120
severe hypertension. Top:
(pg/ml)
100
Blood kinin concentrations 80
before (C) and after n.s.
60
administration of the ACE
40
inhibitor. Bottom: Mean
20
blood pressure before and
after ACE inhibition, open C ACEi
and closed circles represent
rats pretreated with a kinin
200 Veh. or K.ant. Inf.
antagonist or vehicle,
Mean Blood Pressure
respectively. Values are mean

180
SEM (bottom) (reprinted
(mmHg)
from Carbonell et al. [166]) 160
140 P < 0.01

K.ant. (n = 8)
120 Veh (n = 7)
ACEi
5 0 5 10 20 30 40 50
minutes
with a prostaglandin synthesis inhibitor failed to alter the effects of ACE inhibitors,
suggesting that this acute antihypertensive effect is due to bradykinin acting via the
release of NO. However, in the dog, kinins may play a role in the acute hypotensive
effect of ACE inhibitors through the release of prostaglandins [178].
In humans, it has been demonstrated that an ACE insertion (I)/deletion (D)
polymorphism in intron 16 of the ACE gene could be an important determinant
for bradykinin metabolism [179]; ACE activity is higher in subjects with ACE D
and is associated with a high rate of bradykinin degradation. In normotensive sub-
jects and hypertensive patients with low and normal renin, aprotinin (an inhibitor
of kallikrein and other proteases) blocked part of the acute antihypertensive effect
of captopril [180]. The influence of aprotinin could be due to inhibition of kinin
formation or other effects. However, using a specific B2 kinin receptor antagonist
(icatibant), the short-term blood pressure effects of ACE inhibitors were attenuated
in both normotensive and hypertensive subjects [181]. In conclusion, these studies
suggest that part of the acute effect of ACE inhibitors on blood pressure is medi-
ated by kinins, which affect local and peripheral vascular resistance either directly
or through release of prostaglandins and NO.
The contribution of kinins to the chronic antihypertensive effects of ACE
inhibitors is more controversial. In renovascular hypertension (2K1C), chronic
blockade of kinin receptors interferes with the blood pressure-lowering activity
of ramipril [182]. In mineralocorticoid hypertension, in which kallikrein-kinin and
ACE activity are reportedly increased [183], chronic ACE inhibitors have a small but
significant antihypertensive effect; blocking the B2 receptor with icatibant blunted
this chronic antihypertensive action [149, 184], suggesting that in this model kinins
may play a role in the antihypertensive effect of ACE inhibitors. However, they do
not appear to contribute to the chronic antihypertensive effects of ACE inhibitors in
SHR (182] or in hypertension induced by aortic coarctation [165, 185, 186]. There-
fore, the role of kinins in the long-term antihypertensive effect of ACE inhibitors
depends on the model. To our knowledge, no studies of chronic blockade of the
kallikrein-kinin system have been conducted in humans.
12.8 Role of Kinins in the Cardiac Antihypertrophic Effect of

ACE Inhibitors
ACE inhibitors have been shown to reverse LV hypertrophy in essential hyperten-

sion and in various experimental models. This decrease is partly due to reduced
afterload; however, it has been postulated that this antihypertrophic effect may be
independent of the decrease in blood pressure. A decrease in angiotensin II for-
mation, which stimulates various proto-oncogenes and growth factors, may par-
ticipate in the antihypertrophic effect of ACE inhibitors acting independently of
its effect on blood pressure. The cardiac kallikrein-kinin system may also partic-
ipate in the effect of ACE inhibitors on the heart. Doses of ACE inhibitors that
do not decrease blood pressure reverse LV hypertrophy in rats with hypertension
due to aortic coarctation [187]. However, to be certain that blood pressure does
not decrease, direct 24-h measurements are needed. The antihypertrophic effects of
ACE inhibitors have been reported to be reversed by a kinin antagonist [188]. How-
ever, using a very similar protocol, we have not been able to confirm this [186]. Fur-
ther studies are needed to determine whether doses of ACE inhibitors which do not
decrease blood pressure (24-hour blood pressure monitoring) reverse cardiac hyper-
trophy, and whether kinins participate in this effect. Capillary length and density
increase in hearts of SHR treated with an ACE inhibitor at both antihypertensive
and non-antihypertensive doses. There is strong evidence that angiotensin II also
has significant angiogenic effects [189]; however, the effect of an ACE inhibitor
on capillary growth could not be attributed to the inhibition of angiotensin produc-
tion alone, since that effect is blocked by concomitant treatment with a selective B2
receptor antagonist, icatibant, suggesting that this effect of the ACE inhibitor may
be due to kinins [190].
12.9 Role of Kinins in Myocardial Ischemia and in the Protective

Effect of Ischemic Preconditioning and ACE Inhibitors
Both human and animal studies have demonstrated that kinins are released from the
heart and their release is rapidly increased during ischemia. This release of kinins
could have a cardioprotective effect [191193]. Indeed, experimental studies have
shown that intracoronary infusion of bradykinin significantly limited infarct size,
reduced the incidence of ventricular arrhythmias, improved cardiac performance,
and normalized myocardial metabolism [194196]. Tissue kallikrein deficiency
was reported to aggravate cardiac remodeling and survivaval rate after ischemia-
reperfusion injury in mice [197]; whereas B2 / mice or Brown Norway Katholiek
rats (HMWK-deficient) responded similarly to ischemia-reperfusion injury com-
pared to their respective wild types [3], indicating that others than kinins participate
in the cardioaprotective effecst of tissue kallikrein. It has also been suggested that
kinins are an important mediator of ischemic preconditioning, in which repeated
brief coronary occlusions render the myocardium more resistant to injury from
subsequent prolonged ischemia. In patients undergoing angioplasty, balloon infla-
tion for 1 min (which mimics ischemic preconditioning) increased kinin concentra-
tions in the coronary sinus 50-fold compared to pre-inflation values [2]. Precondi-
tioning almost doubled cardiac interstitial kinin concentrations compared to non-
preconditioned hearts subjected to ischemia [193]. The role of kinins in ischemic
preconditioning was further demonstrated in our laboratory using animals geneti-
cally lacking B2 kinin receptors or deficient in kinins. We found that in B2 kinin
receptor knockout mice as well as rats deficient in HMWK, the cardioprotective
effect of preconditioning was abolished or significantly blunted [3].
During myocardial ischemia followed by sympathetic nerve stimulation, kinins
in coronary sinus blood increase significantly [198]. An ACE inhibitor was shown to
reduce myocardial infarct size after ischemia/reperfusion, whereas an angiotensin II
antagonist (losartan) did not [199, 200]. In nephrectomized dogs in which infarc-
tion was induced by occlusion of the coronary artery for 90 min, blockade of
local angiotensin II formation with protease inhibitors had no significant effect on
myocardial infarct size despite decreased angiotensin II release. Captopril did not
alter local angiotensin II formation but did increase bradykinin and reduce infarct
size, suggesting that kinins were responsible for the effect of the ACE inhibitor
on infarct size [192]. Similarly, when low doses of the ACE inhibitor ramiprilat
(which had no systemic effect) were infused into the left coronary artery in dogs,
they reduced the infarction caused by ligation of the descending branch of the left
coronary artery [194]. This cardioprotective effect of ramiprilat was mimicked by
bradykinin and abolished by co-administration of a kinin antagonist.
ACE inhibitors have been shown to reduce ischemia/reperfusion injury, includ-
ing infarct size and reperfusion arrhythmias. Recently, it was recognized that the
cardioprotective effect of ACE inhibitors is due to inhibition of not only angiotensin
II formation but also kinin degradation [4, 5, 199, 201203]. In animal models of
ischemia/reperfusion injury, we and others have shown that ACE inhibitors reduced
infarct size and ventricular arrhythmias and these effects of ACE inhibitors were
abolished or attenuated by co-administration of a B2 kinin antagonist [194, 199,
200, 204]. We further showed that the infarct-limiting and anti-arrhythmia effects
of ACE inhibitors were also blocked by inhibition of NO or prostaglandin synthesis
[200] and diminished in eNOS gene knockout mice [205]. Moreover, deleting the
gene-expressing tissue kallikrein was also associated with decreased cardioprotec-
tive effects of ARB in mice subjected to ischemia-reperfusion injury [206].
The cardioprotective effect of kinins may be mediated in several ways. Release of
NO from the endothelium may be stimulated either directly or via prostaglandins.
It has been shown that myocardial ischemia increases kinin release, accompanied
by increased release of cGMP (an indicator of NO production) and 6-keto-PGF1 (a
metabolite of prostacyclin) [207, 208], whereas inhibiting NO or prostaglandin syn-
thesis diminishes or blocks the cardioprotective effect of kinins [209, 210]. Kinins
improve cardiac metabolism by increasing high-energy phosphate production and
glucogen content in the heart, which could be mediated by facilitating transloca-
tion of intracellular glucose transporters (GLUT1 and GLUT4), thereby increasing
glucose uptake [211, 212]. This is important because during ischemia the source of
energy production is shifted from the oxidation of fatty acids to glycolysis. Also,
activation of protein kinase C (PKC) has been shown to be involved in the protec-
tive mechanism of preconditioning [213215]. Activation of kinins causes further
phosphorylation of a secondary effector, presumably the ATP-sensitive potassium
channels (KATP ). Kinins have been shown to activate PKC, thereby stimulating
opening of KATP and leading to cardioprotection [216, 217]. Such responses may
favorably influence functional and metabolic events during ischemic episodes and
protect against ischemia/reperfusion injury.
12.10 Role of Kinins in the Cardioprotective Effect of ACE

Inhibitors in Heart Failure Post-MI
There is overwhelming evidence that ACE inhibitors reduce morbidity and mortal-
ity, improve cardiac function, regress LV remodeling, and prolong life in patients
with heart failure (HF). We showed that in a rat model of HF due to surgically
Fig. 12.8 Two-dimensional M-mode echocardiographs of B2/ mice and B2+/+ mice with
sham coronary ligation (sham) or HF. IS indicates interventricular septum; DD, LV diastolic dimen-
sion; and PW, LV posterior wall (Modified from [5])
induced myocardial infarction (MI), ACE inhibitors improved cardiac function and
attenuated remodeling, as evidenced by increased ejection fraction and decreased
LV dilatation, myocyte hypertrophy, and interstitial fibrosis. Furthermore, these ben-
eficial cardiac effects of ACE inhibitors were diminished by blockade of kinins
[203]. The role of kinins in the cardioprotective effect of ACE inhibitors was con-
firmed by the fact that in B2 kinin receptor knockout mice (Fig. 12.8) and kininogen-
deficient rats post-MI, the effect of ACE inhibitors was significantly diminished or
absent [5, 202]. Although the precise mechanism by which kinins protect the heart
is not yet well defined, accumulated evidence suggests that kinin-stimulated release
of NO and/or prostaglandins may be largely responsible. Bradykinin stimulates the
release of NO from the mouse myocardium and decreases myocardial oxygen con-
sumption; these effects are blocked by a B2 kinin antagonist and absent in B2 recep-
tor knockout mice [218]. We have shown that the effect of the ACE inhibitor was
almost abolished in endothelial NO synthase (eNOS) knockout mice with HF post-
MI [219]. Taken together, these findings may suggest that kinins acting on the B2
receptor via the release of NO play an important role in the cardioprotective action
of ACE inhibitors. More recently, we have demonstrated that not only B2 but also B1
kinin receptors contribute to the cardiac therapeutic effect of ACE inhibitor [150].
We found that while kinin B1 R does not appear to play an essential role in cardiac
hemodynamics and function either under normal conditions or during development

of heart failure, it may be involved in maintaining morphological integrity, since
mice with targeted deletion of B1 R had increased LV mass and chamber dimen-
sion at basal conditions. Furthermore, the cardioprotective effect of ACE inhibitor
is reduced in mice lacking the gene expressing B1 receptors.
In conclusion, in patients with heart failure, ACE inhibitors have been shown to
not only improve cardiac function and increase survival but also decrease the rate
of myocardial re-infarction [220]. The mechanism of this decrease is not known;
however, since ACE inhibitors may block kinin degradation in the coronary circu-
lation, one hypothesis is that kinins stimulate the release of EDRF and PGI2 , which
are important inhibitors of platelet aggregation. Since kinins are potent stimulators
of the release of tPA [64, 221], it is also possible that this potentiation of tPA release
may in turn activate plasmin and fibrinolysis. Although the exact mechanism of
action of ACE inhibitors in re-infarction is not known, these hypotheses open up an
exciting new area of cardiovascular research.
12.11 Role of Kinins in the Cardioprotective Effect

of Angiotensin Receptor Blockers (ARB)
Two subtypes of angiotensin II (Ang II) receptors, AT1 and AT2 , have been iden-
tified. Most biological actions of Ang II are known to be mediated by the AT1
receptor, whereas little is known about the function of AT2 receptors. In cultured
endothelial cells, Ang II stimulates the release of NO and this effect was blocked
by either an AT2 or B2 receptor antagonist, indicating that Ang II-stimulated NO
release is mediated via activation of the AT2 receptor and a kinin-dependent mech-
anism [222]. Mice overexpressing the AT2 receptor were reported to have increased
kininogenase activity in the vasculature [223]. Since blockade of the AT1 recep-
tor increases angiotensin II levels, which in turn may activate the AT2 receptor, it is
rational to hypothesize that the cardioprotective effect of ARB is mediated in part by
kinins via activation of the AT2 receptor. In fact, we found that ARB improved car-
diac function and ameliorated remodeling in rats with CHF post-MI, and that these
effects of ARB were significantly attenuated by an AT2 or B2 receptor antagonist
[203] or in mice lacking the AT2 receptor [224]. Using B2 kinin receptor knockout
mice and kininogen-deficient rats, as well as eNOS knockout mice, we confirmed
that lack of kinins or endothelium-derived NO diminished the cardioprotective effect
of ARB [4, 5, 219], indicating that increased release of kinins and NO due to acti-
vation of the AT2 receptor is an important mediator of the cardioprotective effect
of ARB. In addition, we recently reported B1 R mediate part of the cardioprotective
effects of ARB in mice with HF post-MI [150].
Because angiotensin II also plays a critical role in the regulation of blood pressure
and in the pathogenesis of many models of hypertension, the interaction of the renin
angiotensin aldosterone system and the kallikrein-kinin system and the contribution
of these two systems on the effects of ACE inhibitors and ARB should not be under-
estimated. Figure 12.9 illustrate some of these interactions. Also, the AT1 receptor
KININOGEN ANGIOTENSINOGEN
RENIN
KININOGENASE ANG I
ACE inh
INACTIVE ( __)
FRAGMENTS \\ KININS ANG 17/III /IV ANG II
ACE inh Cytokines

AT1 ant
B2/1 -receptor AT2/n-receptor
AT1-receptor
iNOS
Ac-SDKP
Aldosterone / Cathechol
NO/EDHF Endothelin / Adhes Molec
Eicosanoids/t-PA Growth Factors / PAI-1 O2 + NO
Pressor/Anti-Natriuretic Cell
Vasodilation/Natriuretic Cardiovascular Remodel Death
Cardiovascular Protection
Liu, Y-H., Yang X-P., and Carretero O.A.

J. Clin. Invest. 1997; 99:19261935
Fig. 12.9 The renin angiotensin and kallikrein-kinin systems. In both systems, a substrate is
cleaved by an enzyme of restricted specificity, releasing a peptide which is either already active
(lys-bradykinin, bradykinin) or inactive (angiotensin I). Upon further processing by a specific
peptidase,angiotensin I is converted to a vasoactive peptide (angiotensin II). In turn, vasoactive
peptides are inactivated by peptidases. Angiotensin-converting enzyme is common to both systems,
but has different roles: it processes angiotensin I to angiotensin II and is the main kinin-inactivating
peptidase (Modified from [1])
and the bradykinin B2 receptor form stable heterodimers, causing increased activa-
tion of G[alpha]q and G[alpha]i (the two major signaling proteins triggered by AT1 ).
Also, the endocytotic pathways of both receptors change with heterodimerization.
This appears to be the first reported example of signal enhancement triggered by
heterodimerization of two different vasoactive hormone receptors [84]. The interac-
tion of the AT1 and B2 receptors potentiates the pressor effect of angiotensin II. On
the other hand, Ang (1-7) interacting with bradykinin has emerged as an endoge-
nous antihypertensive/antitrophic mechanism, opposing many of the Ang II that are
mediated by the AT1 receptor [225227]. It has been demonstrated that Ang (1-7),
acting via receptors other than AT1 or AT2 , induced bradykinin-mediated hypoten-
sion in SHR and normal rats [228] and dilatation of porcine coronary arteries [229,
230]. Ang I and II are cleaved to Ang (1-7) by various endopeptidases [231, 232].
This constitutes another mechanism by which kinins could contribute to the bene-
ficial effects of ACE inhibitors or AT1 antagonists. Bradykinin also appears to play
an important role in mediating the counterregulatory protective effect of AT2 recep-
tors, which oppose the effect of the AT1 receptor [233, 203, 224]. Therefore, there
seems to be a close interaction between kinins and angiotensins in the regulation of
cardiovascular and renal function.
In conclusion, kinins appear not to play a fundamental role in the pathogenesis

of hypertension since in humans, rats, and mice with a deficiency of one compo-
nent of the kallikrein-kinin-system or with chronic blockade of the kallikrein-kinin
system do not have hypertension. In the kidney, kinins participate in the regulation
of papillary blood flow and water and sodium excretion. B2 -KO mice appear to be
more sensitive to the hypertensinogenic effect of salt. Kinins participate in the acute
antihypertensive effect of ACE inhibitors; however, in general, they are not involved
in the chronic antihypertensive effects of ACE inhibitors save for mineralocorticoid-
salt-induced hypertension. Kinins acting via nitric oxide (NO) participate in the vas-
cular protective effect of ACE inhibitors during neointima formation. In myocardial
infarction produced by ischemia/reperfusion, kinins play an important role in the
reduction of infarct size induced by seen after preconditioning or treatment with
ACE inhibitors. In heart failure secondary to infarction, the therapeutic effects of
ACE inhibitors are partially mediated by kinins via the release of NO. The thera-
peutic effect of ARB in heart failure is partly due to activation of angiotensin type
2 receptors, which act via kinins and NO. Thus kinins play an important role in the
regulation of cardiovascular and renal function as well as in many of the beneficial
effects of ACE inhibitors and ARB.
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function. Choices Cardiol 7(Suppl. 1), 1014.
Chapter 13
CMS and Type 2 Diabetes Mellitus:
Bound Together by the Renin Angiotensin
Aldosterone System
Deepashree Gupta, Guido Lastra, Camila Manrique, and James R. Sowers
Abstract The recent epidemic of obesity has led to an increasing incidence of the
cardiometabolic syndrome defined by the NCEP ATP III guidelines as a cluster
of abdominal obesity, low HDL, high triglycerides, HTN, and impaired fasting
glucose. Obesity predisposes the body to a state of inflammation, insulin resis-
tance, and hyperinsulinemia, and individuals with the metabolic syndrome are at
increased risk for developing CAD, stroke, PVD, CKD, and T2DM. There are var-
ious mechanisms by which these complications of the metabolic syndrome occur,
and activation of systemic and local renin angiotensin aldosterone system (RAAS)
and resultant oxidative stress in different organ systems is probably the most impor-
tant one. Finally, based on recent trials, the approach toward management of the
metabolic syndrome is usually multifactorial and multiagent and studies are still
being performed to assess the efficacy of newer drugs.
Keywords Cardiometabolic syndrome Type 2 diabetes mellitus Low HDL High

triglycerides CVD and CKD
13.1 Introduction
Type 2 diabetes mellitus (T2DM) is one of the fastest growing diseases across the
globe and its prevalence has reached epidemic proportions. From 1980 through
2005, the number of Americans with diabetes increased from 5.6 million to 15.8
million, with people aged 65 years or older accounting approximately for 38% of
the population with diabetes (Fig. 13.1). Currently, there are close to 30 million
people in the United States with diabetes and about 8 million of these have not been
D. Gupta (B)
Diabetes and Cardiovascular Center, University of Missouri School of Medicine, and VA Medical
Center, One Hospital Drive, Columbia, MO, USA
e-mail: guptad@health.missouri.edu

188 D. Gupta et al.
Fig. 13.1 Graph representing the increasing prevalence of T2DM in the American population
diagnosed (www.diabetes.org). Obesity, related to both genetic and environmental

factors, and sedentary lifestyle are the leading driving forces behind the dramatic
increase in T2DM in this country. The prevalence of T2DM is on the rise in devel-
oping countries, as well as in industrialized countries including the United States.
Further, the burgeoning burden imposed by diabetes and its complications, including
cardiovascular, renal, and neurological, as well as the medical cost of its treatment
are indeed dramatic.
Risk factors for cardiovascular disease (CVD) and chronic kidney disease (CKD)
tend to cluster with endothelial dysfunction and a litany of metabolic abnormali-
ties [1]. This cluster of metabolic, CVD, and CKD risk factors constitute the car-
diometabolic syndrome (CMS). Key elements of the CMS are abdominal obesity,
insulin resistance, atherogenic dyslipidemia (high LDL, low HDL and hypertriglyc-
eridemia), hypertension (HTN), a prothrombotic and chronic low-grade inflamma-
tory state, CKD, and proteinuria [1] (Table 13.1).
Individuals meeting criteria for diagnosis of the CMS are at increased risk
for developing coronary artery disease (CAD), stroke, peripheral vascular disease
(PVD), and T2DM [1]. About 47 million Americans are currently thought to have
CMS according to the latest American Heart Association (AHA) guidelines [1].
Central to the pathophysiology of the CMS are truncal obesity and insulin resis-
tance (IR) [2]. In this review, we cover potential mechanisms by which IR leads to
the activation of systemic and tissue renin angiotensin aldosterone system (RAAS)
and associated end organ damage [3]. Further, we will discuss various alternatives
that can be used for prevention and treatment. Indeed, with the extremely high preva-
lence of CMS, the prevention of its CVD and CKD complications is of paramount
importance.
13 CMS and Type 2 Diabetes Mellitus 189
Table 1 NCEP ATP III Guidelines for the diagnosis of metabolic syndrome
Risk factor Defining level
Abdominal obesity (waist circumference)

Men >102 cm (>40 in)
Women >88 cm (>35 in)
TG 150 mg/dl
HDL-C
Men <40 mg/dl
Women <50 mg/dl
Blood pressure 130/85 mm Hg
Impaired fasting glucose 110 mg/dl
Diagnosis is established when three or more above-mentioned risk

factors are present.
13.1.1 The Renin Angiotensin Aldosterone System (RAAS)
RAAS plays an essential role in homeostasis of fluids, sodium and potassium. When
the juxtaglomerular cells (JG) of the kidney are stimulated by decreased renal per-
fusion or decreased sodium content in blood or increased beta 1 adrenergic activ-
ity, they become activated and stimulate the production of rennin [3]. Renin is a
protease that processes angiotensinogen in the liver, which results in the formation
of the decapeptide angiotensin I. Angiotensin I is in turn converted by angiotensin-
converting enzyme (ACE) to the active octapeptide angiotensin II (Ang II) mainly in
the pulmonary circulation by removing two amino acids from the carboxy terminus
of angiotensin I. ACE is a membrane-bound enzyme anchored to the endothelium of
many vascular beds, with the highest concentrations found on the vascular epithe-
lium of the lung. Angiotensin II (Ang II) in turn stimulates the production and secre-
tion of aldosterone from the zona glomerulosa of the adrenal gland. Aldosterone is
a mineralocorticoid that acts by signaling through the mineralocorticoid receptor
(MR) to increase tubular reabsorption of sodium and thus salt and water retention
with attendant increases blood pressure [3]. Ang II, which is a potent vasoconstric-
tor, also contributes to the elevation in blood pressure and, working in concert with
aldosterone, promotes tissue remodeling and injury [3]. The effects of Angiotensin
II are mediated through two primary receptors AT1 R and AT2 R. AT1 R has two
subtypes AT1 A and AT1 B, but most of the pressor, growth, and tissue remodeling
effects of Ang II are mediated via the AT1 A receptor.
In addition to circulating RAAS, there is accumulating evidence supporting
the importance of local RAAS in several tissues. These have been described in
the brain, kidney, adrenal, testis, and arterial wall in animals and in humans
[2]. As angiotensinogen is the precursor of angiotensin peptides, local synthe-
sis of angiotensinogen is required to demonstrate the existence of a local RAAS.
Angiotensinogen mRNA has been described in several different extrahepatic tissues
in mice, including kidney, brain, spinal cord, aorta, mesentery, adrenal, atria, lung,
190 D. Gupta et al.
stomach, large intestine, spleen, and ovary. Renin mRNA has also been detected in
many extrarenal tissues (adrenal, heart, testes, and submaxillary gland) [3].
Although RAAS is important in maintaining homeostasis in the body, inappro-
priate activation of circulating and/or local RAAS can trigger excessive oxidative
stress in numerous tissues. Increased RAAS activity in the CMS is related to abdom-
inal/truncal obesity [4] (obesity per se is a salt-retaining and volume expansion
state), fatty acid (FA) elevation [5], insulin resistance (IR)/compensatory hyperinsu-
linemia, HTN, hyperglycemia [6] and atherogenic dyslipidemia. Indeed, a local RAS
system [5] has been well delineated in adipose tissue, and this local RAS plays an
important role in adipocyte differentiation, as well as oxidative stress, inflammation,
and the production and secretion of adipocyte cytokines.
13.1.2 The Role of Adipose Tissue and Adipokines
Adipose tissue is an active endocrine organ responsible for the production several
adipokines, which have numerous autocrine, paracrine, and endocrine actions. In
obesity and the CMS, the adipose tissue is dysfunctional and is characterized by
an imbalance between pro-inflammatory and insulin-sensitizing adipokines, which
contribute to the development of a chronic low-grade inflammatory environment and
insulin resistance [7]. There is increased secretion of FA originating in dysfunctional
adipose tissue, leading to increased hepatic gluconeogenesis and impaired intracel-
lular insulin signaling. IR in turn results in increased lipolysis and further increase
in FA levels creating a vicious cycle [8].
Resistin is produced in white adipose tissue in mature adipocytes as well as dur-
ing differentiation of adipocytes. Under experimental conditions its levels decline
with prolonged fasting and increase with food intake and when injected into normal
mice, it can induce hepatic and skeletal muscle insulin resistance [9, 10]. However,
animal and human trials have yielded controversial results, and additional studies
are needed to ascertain the role of this hormone in the pathophysiology of IR and
CMS.
TNF-, mainly through a paracrine effect, causes elevation of FA levels and
inhibits tyrosine phosphorylation of Insulin Receptor Substrate (IRS-1), which is
required for insulin intracellular signal transduction; it does this by promoting serine
phosphorylation of IRS-1. In addition, TNF- contributes to activation and persis-
tence of chronic inflammation in the adipose tissue by activation of Th1 lympho-
cyte subpopulation, increased production of monocyte chemotactic protein 1 (MCP-
1), monocyte colony-stimulating factor (M-CSF) in activated macrophages as well
as superoxide dismutase and proinflammatory interleukins (particularly IL-1 and
IL-6). TNF- also activates the nuclear factor kB (NF-kB) pathway in endothelial
and vascular smooth muscle cells (VSMCs), hence promoting endothelial dysfunc-
tion and eventually atherogenesis [11, 12]. Finally, the insulin-sensitizing adipokine,
adiponectin, is inhibited by TNF- [13].
IL-6, unlike TNF-, is found in high levels in blood and has mainly an endocrine
action. Levels in blood are directly proportional to BMI, insulin resistance, and
impaired glucose tolerance [14, 15]; conversely, weight loss leads to reduction in
circulating and adipose levels of IL-6 [9]. IL-6 causes insulin resistance, hyper-
glycemia, and dyslipidemia via the expression of SUPPRESSOR of cytokines 3
(SOCS-3), which impairs intracellular transduction of both insulin and leptin [14].
Similar to TNF-, IL-6 inhibits adiponectin secretion and action [15].
Leptin is another adipokine implicated in the pathophysiology of the CMS. It is
produced by adipose tissue and its circulating levels are proportional to the fat body
mass [16]. Leptin acts on the arcuate nucleus of the hypothalamus and decreases
appetite in concert with increasing energy expenditure, hence resulting in decreased
adipose mass and weight loss [17]. When injected into the CNS of mice geneti-
cally unable to produce it (Ob/Ob mice), leptin reverts hyperglycemia and hyper-
insulinemia, most likely through activation of adrenergic pathways [18, 19]. Leptin
also acts on peripheral tissues like skeletal muscle where it improves insulin sensi-
tivity. It has been proposed that this action is via 5 AMP-activated protein kinase
(AMPK) pathway, which inhibits anabolic processes involving consumption of ATP
and stimulates catabolic pathways like cellular glucose transport and glucose and
FA oxidation that produce ATP, the net effect being energy expenditure and reduc-
tion of energy stores, i.e., adipose tissue mass [2022]. It has been demonstrated
that selective leptin resistance and hyperleptinemia result in inability to lose weight
and preservation of sympathoneural outflow and sodium resorption ability, which
contribute to both HTN and obesity in the CMS.
Finally, adiponectin is an insulin-sensitizing and anti-inflammatory adipokine,
which as has been mentioned above, is inhibited by both TNF- and IL-6, more so
in obesity and dysfunctional adipose tissue. The mechanism of action of this peptide
remains to be fully uncovered, but it is thought to implicate FA oxidation in skele-
tal muscle by stimulating the AMPK activity and decreasing the hepatic glucose
output [23]. It also prevents endothelial damage and has antiatherogenic activity,
through inhibition of the expression of adhesion molecules like ICAM-1, VCAM-
1, E-selectin, andTNF- [24, 25]. Some studies have shown that adiponectin sup-
presses macrophage migration and their transformation into foam cells, decreases
vascular intimal proliferation and via AMPK stimulation induces NO synthesis [25].
13.1.3 Hyperinsulinemia in CMS
It is well known that in obesity, glucose uptake to peripheral tissues, especially to

skeletal muscle, in response to insulin via GLUT-2 and GLUT-4 receptors becomes
impaired. IR and compensatory hyperinsulinemia (HI) impair the homeostatic
actions of insulin and results in enhanced activity of the sympathetic nervous sys-
tem, increased sodium and water reabsorption in proximal renal tubules, decreased
urinary sodium excretion, activated systemic RAAS, increased arterial tone and
pressure by increased membrane transport of calcium, and increased number of
192 D. Gupta et al.
AT1 R. IR also stimulates vascular smooth muscle cell proliferation, migration, and
vascular extracellular matrix remodeling [26, 27]. It is thought to do so by the acti-
vation of local RAAS and via adverse effects of Ang II, which are oxidative stress
from reactive oxygen species (ROS) (discussed in detail later), impaired insulin
metabolic pathway (P13K/Akt), and increased proliferative and remodeling path-
ways (MAP kinase pathway). All these intracellular and intravascular changes in
IR lead to increased blood volume and HTN [28]. In fact, the existence of insulin
resistance and/or compensatory hyperinsulinemia in patients with essential HTN has
been extensively documented [29], and hyperinsulinemia as a surrogate of insulin
resistance has also been shown to be an independent predictor of HTN [3033]. IR
and/or compensatory hyperinsulinemia have also been shown to predict T2DM in
several prospective studies [3437].
13.1.4 HTN and The CMS
HTN is a condition of multifactorial origin, in which genetic predisposition is a

predominant feature. The European Prospective Investigation into Cancer Norfolk
(EPIC) study in which more than 20,000 participants were included showed that
both systolic and diastolic blood pressures increased in a directly proportional man-
ner to the waist-to-hip ratio in men as well as women [38]. The mechanisms involved
in obesity-related HTN are complex and involve derangements in multiple systems.
They include activation of the RAAS, increased sympathetic nervous system (SNS)
activity, insulin resistance, increased renal sodium reabsorption, impaired pressure
natriuresis, and vascular volume expansion. In addition, obesity may also cause
marked structural changes in the kidneys, which will eventually lead to CKD and
further increases in blood pressure [28, 39, 40].
A direct correlation has been demonstrated between blood pressure and blood
glucose levels in essential HTN. Uncontrolled HTN is associated with both elevated
fasting and post prandial blood glucose levels [41]. It has been proposed that about
50% of patients with essential HTN will develop T2DM over 1015years [4244].
13.1.5 Dyslipidemia
Atherogenic dyslipidemia consisting of high triglycerides (TGs) and low-density

lipoprotein cholesterol (LDL-C) and low high-density cholesterol (HDL-C) is
closely associated with obesity. In addition, elevated TG/HDL-C ratio is associated
with elevated blood pressure and is also an independent risk factor for IR [45].
13.2 Oxidative Stress

There are various systems in the mammalian cells that can produce ROS and cause
excessive oxidative stress. These include the NADPH oxidase enzymatic complex,
nitric oxide synthase, cytochrome p450 enzymatic complex, the mitochondrial elec-
tron transport system, and the xanthine oxydase system [46]. Most of the work done
on animal models implicates the NADPH oxidase system in causing the maximum
oxidative stress-induced tissue damage in studied tissues such as cardiovascular,
renal, and skeletal muscle.
The NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) is a
membrane-bound electron transport complex that catalyzes the production of super-
oxide from oxygen and NADPH. It is a multisubunit enzyme composed of three
cytosolic (p40phox , p47phox and p67phox ) and two membrane-bound components
(Nox 2 (P91phox ) and p22phox ) plus the small proteins Rac 1 and Rac 2, which are
essential to NADPH oxidase assembly. Phox stands for phagocytic oxidase and
Rac 1 or Rac 2 (Rac stands for Rho-related C3 botulinum toxin substrate) is a Rho
guanosine triphosphatase (GTPase).
Activation of the oxidase involves the assembly in the plasma membrane of mem-
brane bound and cytosolic components of the NADPH oxidase system, which are
disassembled in the resting state. Activation starts with the phosphorylation of one
of the cytosolic components and their translocation to the plasma membrane where
electron transfer between gp 91 and O2 molecules leads to the formation of super-
oxide (O2- ) and ROS [47].
Ang II via AT1 R can promote this pathway by stimulating intracellular pathways
that result in translocation of cytosolic subunits to the plasma membrane, direct
phosphorylation of membrane bound subunit p22 phox via PKC activation and acti-
vation of Rac 1 by association with caveolin 1 [48, 49].
ROS cause multiple structural and functional changes in numerous tissues, and
in particular to endothelial cells. ROS can induce the inflammatory NF-kB path-
way and hence increase the expression of Vascular Adhesion Molecule 1(VCAM1)
[50]. They can also activate tyrosine kinase pathways like extracellular signal-
regulated kinase 1 and 2 (ERK1 and ERK2) and cause transactivation of growth
factor receptors like EGFR; the end results of these pathways being influence
on vascular cell growth and proliferation. ROS can also trigger the Jak-STAT
pathway leading to increased IL-6 production, which as mentioned above causes
inflammation [51].
The gap in our knowledge between local RAAS activation, oxidative stress, and
insulin resistance is bridged in the adipose tissue. As described in detail above,
local RAAS activates adipokines in the adipose tissue contributing to insulin resis-
tance, which in turn leads to chronic inflammation. Current research indicates that
angiotensinogen messenger RNA expression is higher in abdominal fat compared to
subcutaneous fat, a finding that correlates with the differences observed in insulin
resistance between the two tissues [52].
194 D. Gupta et al.
13.3 Effects of RAAS on Various Organs

Local RAAS is thought to be responsible for the production of reactive oxygen
species and endothelial dysfunction, which in turn cause end organ damage in mul-
tiple organs. In addition, Ang II is directly responsible for structural modification of
organs like the heart. In the next section, we will try to describe the various changes
taking place at tissue levels in the different end organs involved in the pathophysi-
ology of the CMS and T2DM.
13.3.1 Heart
Both insulin and IGF-1 via their receptors in cardiovascular tissue (53) result in
vasorelaxation (54) and myocardial glucose uptake (41, 5557). Ang II receptors
have been characterized in cardiomyocytes and cardiac fibroblasts as well as in the
endothelial lining of coronary arteries. Ang II, via e AT1 R, and especially AT1A R,
causes peripheral vasoconstriction, increasing the peripheral vascular resistance and
hence maintains blood pressure in the face of decreased cardiac output (58). Ang II,
in addition to its vasoconstriction effects, attenuates the metabolic actions of insulin
and IGF-1 in cardiovascular tissue, via the generation of ROS and activation of small
molecular weight proteins such as RhoA and Rac1 (41, 53, 59). It also stimulates
the release of cathecholamines from noradrenergic nerve endings and salt and water
retention via aldosterone synthesis from the adrenal gland.
Many of the detrimental effects of both Ang II and aldosterone are mediated
via the activation of membrane NADPH oxidase as well as mitochondria-generated
ROS. This Ang II-induced oxidative stress affects cell signaling responses and facil-
itates marked cardiac hypertrophy, interstitial fibrosis, and left ventricular dysfunc-
tion [58, 60, 69].
13.3.2 Endothelium
Insulin/IGF-1 stimulates PI3K/PDK-1/Akt phosphorylation of human eNOS at

Ser1177, resulting in enhanced eNOS; eNOS in turn mediates endothelial cell
production of NO. Insulin and IGF-1 also increase vascular smooth muscle cell
(VSMC) production of NO and attenuate Ang II-induced increase in cytosolic cal-
cium and myosin light chain (MLC) kinase activity; which result in vascular relax-
ation [58].
Ang II promotes vascular growth/remodeling, apoptosis, and fibrosis via
increased generation of ROS. These markedly reactive ROS molecules oxidize
lipids, protein, and DNA and cause cellular injury. They cause vasoconstriction by
converting NO to peroxynitrite (ONOO- ). ROS activate the transcription of factors
such as TNF-, monocyte chemoattractant protein (MCP)-1, IL-6, and C-reactive
protein (CRP), and TNF- impedes insulin- and IGF-1-mediated eNOS activation
as well as their antiapoptotic actions [58].
Recently, new studies have reported insulin receptor expression in macrophages
and implicate protein CD36 to play an important role in atherogenesis [61]. Protein
CD 36 is a glycoprotein in the platelet membrane and also a class B scavenger recep-
tor on macrophages that plays an important role in recognition, uptake, and inter-
nalization of oxidized LDL molecules in the early steps of atherogenesis [62, 63].
In obese, insulin-resistant Ob/Ob mice models, there is an increase in oxidized LDL
uptake in macrophages and this event is associated with a simultaneous increase in
CD36 concentrations. Increased CD36 concentrations adversely affected the tyro-
sine kinase activity of the insulin receptor and intracellular insulin signaling, further
contributing to insulin resistance [63, 64].
13.3.3 Kidney
As previously discussed, renin stimulates the synthesis of Ang II and aldosterone,

which in turn acts on the mineralocorticoid receptor in the collecting duct, lead-
ing to salt and water retention. Obesity and IR/hyperinsulinemia have been shown
to have deleterious effects on the renal hemodynamics, causing reduced-pressure
natriuresis and increased salt retention. These changes in turn result in hyperfiltra-
tion and increased glomerular filtration rate (GFR, andurinary albumin excretion
[65] in experimental conditions [66] as well as hypertensive individuals [67].
The effects of inappropriately activated RAAS on the kidney have been exten-
sively studied in the TG (mRen2)27 (Ren2) transgenic rat model, which overex-
press the mouse renin gene and exhibit increased tissue Ang II levels in glomerular
mesangial cells. Activation of AT1 R by Ang II increases oxidative stress, inflamma-
tion, and endothelial dysfunction which in turn cause HTN, glomerular injury, loss
of filtration barrier, and albuminuria. In the Ren2 rat model, electron microscopy
(EM) measurements have demonstrated podocyte foot process effacement, loss of
slit pore diaphragm integrity, and widening of the bases of the podocyte foot process
[45]. The podocyte, which is the most differentiated cell type within the glomeru-
lar complex, is an integral component of glomerular basement membrane and slit
pore diaphragm and hence plays a pivotal role in maintaining glomerular filtration
barrier. Podocyte injury therefore causes destabilization of the foot process/slit pore
diaphragm complex, with resultant loss of glomerular permeability leading to albu-
minuria. These structural changes are associated with increased NADPH oxidase
activity in renal cortical tissue [45].
T2 DM and HTN are the most common causes of end-stage renal disease
[6870]. The earliest clinical manifestation of nephropathy is the presence of
microalbuminuria (MAU), defined as urine albumin of 30300 mg/day or 30
300 mg/g of creatinine in a spot urine collection. MAU has been integrated as a
diagnostic criterion in the WHO criteria for definition of the CMS [71, 72]. MAU is
considered to be an early marker of endothelial dysfunction and kidney impairment,
196 D. Gupta et al.
and its presence heralds the progressive loss of renal function [7375]. In addi-
tion, it is considered an independent risk factor for the development of CVD in this
patient group. This could probably be explained by the fact that elevated BP and
poor glycemic control in T2DM that contribute to MAU are both associated with
CVD and CKD [28]. ACEI and ARBs decreased MAU as well as overt proteinuria
in addition to their other effects, and are the frontline medications used in HTN and
proteinuria management in patients with T2DM.
13.3.4 Pancreas
It has recently been identified that the pancreatic islet beta cell expresses pro-renin,
renin, Ang II, and AT1 R [76, 77]. It is thought that the physiological role of the
pancreatic RAS in mice models seems to involve islet blood flow regulation, which
would affect glucose-stimulated insulin secretion and homeostasis of carbohydrate
and fat metabolism [78]. The Ren2 rat model has increased islet Ang II and AT1 R.
This activation of local tissue RAS in the rat pancreas leads to NADPH oxidase-
mediated generation of ROS. Pancreatic islets are highly vulnerable to oxidative
stress since they have a low intrinsic antioxidant capacity [79, 80]. Changes induced
by excessive oxidative stress include disordered islet architecture, increased fibro-
sis at the islet-exocrine interface, pericapillary fibrosis, and increased structurally
abnormal mitochondria in both the endocrine and the exocrine pancreas. These
changes are associated with significantly impaired islet blood flow and decreased
insulin release from mouse islets in response to high glucose; thus oxidative stress
may play an important role in the early stages of insulin resistance and T2DM
[79, 81]. Ang II-mediated increased levels of NADPH oxidase in the islets is thought
to stimulate the mitochondria to generate ROS via the citrate synthase and electron
transport chain. This could explain the increased numbers of structurally abnormal
mitochondria seen in the endocrine and exocrine pancreas as described above [80,
82, 83]. One of the ROS is superoxide, which by activating uncoupling protein 2
(UCP2), diverts energy away from ATP synthesis and hence decreases the ATP/ADP
ratio. This in turn leads to less efficient glucose-dependent insulin secretion from the
pancreas [82, 83]. Superoxide can also adversely affect cell neogenesis as it irre-
versibly decreases one of their important transcription factors PDX-1 [80]. ROS,
by increasing the IRS-1 serine (Ser) phosphorylation, leads to proteosomal degra-
dation. Oxidative stress in the islet triggers endoplasmic stress [84], which causes
more Ser phosphorylation of IRS-1 and further contributes to cell dysfunction
[85].
In the exocrine pancreas, angiotensinogen, AT1 R, and AT2 R are localized in the
pancreatic ducts, blood vessels, and acinar cells [2, 76, 86, 87]. This local RAS
system appears to regulate the pancreatic microcirculation, acinar enzyme secretion,
and pancreatic pericyte and stellate cell function [8689].
Oxidative stress can cause pancreatic exocrine inflammation and fibrosis in an
experimental model of pancreatitis. These in vivo treatment with aliskiren, a direct
renin inhibitor, normalized systemic insulin resistance and islet insulin, decreased
islet Ang II, NADPH oxidase activity/subunits and nitrotyrosine, and improved total
IRS-1 and Akt phosphorylation as well as islet/exocrine structural abnormalities
[90].
13.3.5 Adipose Tissue
Ang II, via AT1 R activation, inhibits preadipocytes differentiation into adipocytes.
Decreased capacity of adipose tissue to store FA can lead to FA deposi-
tion/accumulation in other tissues including skeletal muscle and liver, leading to
insulin resistance and contributing to the development of T2DM [8, 14, 91]. In addi-
tion, dysfunctional adipose tissue produces proinflammatory cytokines such IL-1,
TNF-, and resistin, which in turn induces synthesis and release of chemoattractant
factors in the stromal adipose tissue. These factors mediate macrophage infiltration
of adipose tissue, endothelial dysfunction, and atherogenesis. Thus, adipose tissue
plays a role as both cause and target of a low-grade inflammatory state and pro-
vides a direct relationship to atherosclerosis, leading to generalized vascular dam-
age, HTN, and CVD [92].
13.3.6 Therapeutic Approach for CMS
The treatment of the CMS and its individual components require a multifactorial
intervention. Insulin resistance and impaired fasting glucose/impaired glucose tol-
erance (termed prediabetes by some authors) predisposes a person to developing
T2DM in the next 10 years and increases their risk for CVD [93, 94].
Therapeutic lifestyle modifications are the cornerstone of the multifactorial ther-
apeutic approach to the CMS. Of paramount importance are a balanced low-fat
diet, light to moderate regular exercise, and smoking cessation. From a pharma-
cologic standpoint, use of aspirin, reduction of blood pressure, reduction of gly-
cated hemoglobin to less than 6.5% in diabetics, and control of hyperlipidemia have
proven their efficacy. After a mean follow-up of 7.8 years, this approach results in
up to 20% reduction in the risk of CVD [95]. The reduction in CVD risk is higher
compared with previous trials targeted at control of isolated risk factors like hyper-
glycemia, HTN or dyslipidemia, mainly by means of pharmacological intervention
[9698].
As previously discussed, HTN increases the risk of T2DM in the next 10 years.
Antihypertensives that produce RAAS blockade, in particular ACEIs and ARBs,
have been related to a reduced incidence of T2DM. The Prospective Randomized
Open Blinded End point (PROBE) trial, which compared the effect of captopril
versus conventional therapy (beta-blockers and diuretics) on CVD morbidity and
mortality, failed to demonstrate a significant difference in overall CVD morbidity
and mortality in the ACEI-treated group, but a significant reduction of 30% in the
198 D. Gupta et al.
incidence of DM2 was observed after a follow-up of 6.1 years [99]. The PROBE
study suggests that RAAS inhibition is particularly beneficial in patients with DM2,
and that the reduction observed in the incidence of DM2 could lead to a further
reduction of CVD in the long term.
The HOPE trial also demonstrated similar outcomes with the incidence of new-
onset diabetes being 34% lower in the ramipril-treated group as compared to placebo
[100]. Other studies that have demonstrated decreased incidence of new onset-
diabetes with the use of ACE inhibitors and Ang II-receptor blockers include
ALLHAT [101, 102], SOLVD [103], and LIFE [104]. The CHARM-Added trial
demonstrated that the combination of ACEIs and ARBs was associated with a
more profound reduction in the incidence of T2DM as compared with the use of
either agent alone [105]. ACEIs have been demonstrated to delay the progression
of microalbuminuria in T2DM patients without HTN, probably through blood pres-
sure control, reduction in intraglomerular pressure, control of mesangial prolifera-
tion and of transmembrane protein leakage. Importantly however, in these studies,
the primary outcome was not the effect of ACEIs and ARBs on the incidence of
DM2, but cardiovascular outcomes.
On the other hand, the Diabetes Reduction Assessment with Ramipril and
Rosiglitazone Medication (DREAM) trial, which actually targeted the incidence of
T2DM, demonstrated that the use of ramipril was not associated with a significant
reduction in the incidence of T2DM but resulted in significantly increased regres-
sion to normoglycemia relative to placebo, thus suggesting a beneficial effect of
RAAS blockade on glucose homeostasis [106].
Possible mechanisms responsible for the reduced incidence of diabetes in these
trials include improved insulin sensitivity, enhanced endothelial function, increased
nitric oxide activation, reduced inflammatory response, and increased bradykinin
levels [107]. A recent study has shown that treatment with low dose of losar-
tan (50 mg) significantly elevated the serum concentrations of total adiponectin
in patients with essential hypertension [108]. However, in the previously diabetic
group, captopril was associated with a reduced incidence of fatal and nonfatal CVD
[109].
In numerous studies, statins have been reported to reduce the incidence of T2DM.
The Heart Protection Study (HPS) [110] showed that simvastatin 40 mg/d signifi-
cantly decreased (approximately 25%) major cardiovascular events in persons with
T2DM relative to placebo regardless of the baseline LDL-C level. These findings
were reproduced in the Collaborative Atorvastatin Diabetes Study (CARDS) [111]
and the Atorvastatin Study for Prevention of Coronary Heart Disease Endpoints in
Non-insulin-Dependent Diabetes Mellitus (ASPEN) [112]. A step further were the
CORALL [113] and Pravastatin or Atorvastatin Evaluation and Infection Therapy
(PROVE IT) [114] studies, which showed that high-dose statins and more aggressive
LDL-C lowering were more effective in lowering the incidence of CVD. However,
it is thought that the benefits of statins go beyond their LDL-C lowering action
and include improvement of endothelial function and anti-inflammatory actions
by inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.
The inhibition of this enzyme in turn causes decreased isoprenylation of signaling
molecules like Ras, Rho, and Rac, which are involved in smooth muscle prolifera-
tion, inflammation, oxidative stress, and cellular remodeling [115].
Dyslipidemia contributes to the progression of glomerulosclerosis and diabetic
nephropathy [116, 117]. Treatment with statin therapy reduces MAU and delays
the progression of diabetic nephropathy [6870, 118120] in experimental ani-
mals [121, 122] and in diabetic patients [123, 124]. Experimental data suggest that
statins do this by reducing mesangial cell proliferation and fibrogenesis and also by
decreasing the generation of ROS, macrophages, and inflammatory cytokines [121,
122, 125]. Statins have also been shown to reduce podocyte injury and effacement
[121, 125]; both these processes are thought to be involved in the development of
proteinuria. These effects can be explained by the fact that podocytes have lipopro-
tein receptors, and that these receptors are highly expressed in proteinuric states
[126].
As the RAAS has been found to be the culprit in inducing oxidative stress in
various organ systems, medications that block this system at various levels are being
studied in detail. In addition to the already discussed role of ACEIs and ARBs,
aliskiren is being used to control HTN and has been seen to reverse the effects of
ROS in rat pancreas.
On the other hand, hyperaldosteronism is also associated with insulin resistance.
The underlying mechanisms leading to this impaired insulin sensitivity remain to
be fully elucidated, but involve increased production of ROS and oxidative stress.
It has recently been found that mineralocorticoid receptor (MR) antagonism can
reduce oxidative stress and improve insulin sensitivity in skeletal muscle in Ren2
rats independently of any effect on blood pressure [127].
In human clinical studies, Catena et al. confirmed increased insulin resistance as
well as impaired glucose utilization in primary aldosteronism (PA) patients but not
in essential hypertensive individuals [128]. Fallo et al. recently reported a 41.1%
prevalence of CMS in PA patients compared to 29.6% in essential hypertensives
(p < 0.05), again underscoring the fact that hyperaldosteronism is an independent
cause of insulin resistance [129].
The RALES and EPHESUS trials have demonstrated beneficial effects of
spironolactone in decreasing cardiovascular morbidity and mortality. The RALES
(Randomized Aldactone Evaluation Study) was a double-blind trial, which included
patients who had severe heart failure and left ventricular ejection fraction below
35%. It was seen that participants who received 25 mg of spironolactone vs placebo
plus conventional treatment with an ACEI, a loop diuretic, and in most cases digoxin
had a 35% reduction in the relative risk of death; this was attributed to a lower risk
of both death from refractory heart failure and sudden cardiovascular death. Patients
who received spironolactone also had a significant improvement in the symptoms of
heart failure [109]. In the EPHESUS (Eplerenone PostAcute Myocardial Infarction
Heart Failure Efficacy and Survival Study) trial, there was a significant reduction
in cardiovascular mortality and the rate of death from any cause or hospitaliza-
tion among patients receiving eplerenone [109]. These effects could be due to MR
blockers action against HTN, oxidative stress, inflammation, apoptosis, and fibrosis
in the cardiovascular and renal tissue [130].
200 D. Gupta et al.
To summarize, the demonstration of cardiovascular injury and impaired insulin

signaling in the setting of increased aldosterone as well as clinical studies showing
improvement in HTN and glucose homeostasis through pharmacological blockade
of the MR support a direct correlation between mineralocorticoids and CMS. The
mechanisms underlying these relationships still remain to be fully elucidated; how-
ever, RAAS-mediated increased oxidative stress appears to play a key role.
13.3.7 Conclusions and Perspectives
The cluster of HTN, T2DM, HLD, and MAU define that the CMS is growing dra-
matically, driven in large part by the excess weight. Obesity results in dysfunctional
adipose tissue, chronic low-grade inflammation, insulin resistance, and hyperinsu-
linemia, which as demonstrated above, activate systemic and local RAAS in diverse
tissues such as heart, endothelium, kidneys, pancreas, and adipose tissue. Local
RAAS activation is associated with the production of ROS and oxidative stress,
which has various deleterious effects on the morphology and function of these organ
systems, leading to T2DM, CKD, atherogenesis, and CVD.
A multifactorial approach has been recommended for the management of CMS
with diet, exercise, and weight loss still being the therapeutic cornerstone. There is
also consistent evidence in numerous experimental and clinical studies demonstrat-
ing a paramount role of inappropriate activation of RAAS, subsequent increased
oxidative stress and insulin resistance in the pathophysiology of the CMS, and devel-
opment of T2DM. Different strategies to block RAAS, in particular the use of ACEIs
and ARBs, have emerged as important alternatives, which are now used ubiquitously
to prevent complications of CMS, have shown benefits in terms of CVD morbidity
and mortality. In addition, there is promising evidence about drugs like aliskiren,
statins and MR antagonists. These agents block the RAAS at different levels, and
statins are thought to ameliorate the oxidative stress induced by insulin resistance.
The multifactorial approach to the management of CMS, in concert with exciting
new research, will contribute to the prevention and management of the CMS and to
reduce its burden on healthcare worldwide.
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Chapter 14
Renin Angiotensin Aldosterone System and
Cardiovascular Disease
Swynghedauw Bernard, Milliez Paul, Messaoudi Smail, Benard Ludovic,

Samuel Jane-Lise, and Delcayre Claude
Abstract Aldosterone, aldo, is a rather minor component of the adrenal gland

production with complex activity. Aldo and glucocorticoid are in competition and
the specificity of aldo action is due to a cellular component the 11-HSD2. Aldo
at high concentrations has pronounced and deleterious cardiovascular effects and
causes pro-inflammatory reaction followed by myocardial and vascular fibrosis.
Fibrosis modifies cardiac performances and has proarrhythmogenic consequences
and is prevented by spironolactone and/or eplerenone. At low concentrations
aldo inhibits BKCa potassium channel and causes coronary dysfunction with-
out major alterations in myocardial function. Besides its classical mode of action
on MR, aldo modulates intracellular Ca and cAMP concentrations, phospho-
rylates several kinases of major importance, and activates the EGFR signaling
pathways. Aldo and angiotensin II are partners; on one hand, aldo is able to
activate the transcription of components of angiotensin II activity; on the other
hand, the angiotensin II-dependent increase in collagen is in part dependent
on aldo.
Keywords Aldosterone Angiotensin II Glucocorticoid Eplerenone

Spironolactone Transgenic mice Aldosynthase
14.1 Introduction
The RALES [1] and EPHESUS [2] clinical studies have demonstrated the impor-
tant benefit of MR antagonists in patients with heart failure or with left ventricular
dysfunction after myocardial infarction. A short-minded conclusion would there-
fore be that aldosterone plays a generally evil role and that it is important to block it
in all circumstances. This is obviously not so simple, and to better understand its
S. Bernard (B)
Centre de Recherches Cardiovasculaires INSERM Lariboisire, PARIS, France
e-mail: bernard.swynghedauw@inserm.fr

208 S. Bernard et al.
mechanisms and better anticipate the effects of treatment, several points should
be highlighted. The classical role of aldosterone is to adjust the hydro-mineral
balance in the body, and thus to decisively intervene in blood pressure control.
Meanwhile, experimental studies have demonstrated that aldosterone induces struc-
tural and functional changes in the heart, kidneys, and blood vessels, with pro-
nounced cardiac and renal fibrosis, inflammation, vascular remodeling, and changes
in fibrinolysis. These damages are said to be mediated by aldosterone and are pre-
vented or minimized by MR antagonists as spironolactone or eplerenone. It can-
not be excluded that potassium and hypertension may also play a key role in these
damages. Nevertheless above all, it is important to stress that these effects were
observed at very high aldosterone concentrations, too high to complain for the body
salt requirements [3]. In addition, a distinction must be made between inhibition of
MR and antagonism of the effects of aldosterone. Indeed, the presence of gluco-
corticoids in concentrations much higher than those of aldosterone in plasma, the
structural similarity of these two hormones and of their receptors, the hormone
receptor affinities measured in vitro that conclude to the possible binding of cortisol
(or corticosterone in rodents) on the aldosterone receptor (and vice versa) compli-
cate the understanding of the mode of action of aldosterone in the cardiovascular
system.
14.2 Biosynthesis of Aldosterone
A substance secreted by the adrenal glands and having the ability to retain salt
has been evidenced for the first time in the 1930s by the teams of Kendall and
Reichstein (for an historical review on the discovery of aldosterone, see [4]). From
the crystallization of glucocorticoids and mineralocorticoids, researchers have dis-
covered that a part of the fraction extracted from adrenals was not crystallized.
This fraction called amorphous had an important mineralocorticoid activity, albeit
different from that of deoxycorticosterone or other steroids. It was not until the
1950s and the improvement of biochemical techniques that some researchers (con-
vinced of the existence of a mineralocorticoid different from desoxycorticosterone)
were interested in this fraction. Advances in the techniques for determining the
sodium/potassium ratio using radioactive compounds, and of chromatography from
urine of adrenalectomized rats or from extracts of beef adrenal gland, have finally
allowed to purify the active compound. This active fraction, first called electro-
cortin because of its properties on the electrolytes metabolism, will then be crys-
tallized. The hormone was then better characterized under the name of aldosterone.
That this hormone was secreted by the adrenal gland was then evidenced by Taits
team, showing that the hormone extracted from beef or dog adrenal perfusate
were identical. Such a rapid retrospective highlights the opposition that existed
between the supporters of cortisol seen as the genuine adrenal hormone (aldos-
terone being an artifact of the synergistic action of steroids), and the supporters of
aldosterone who thought it was a hormone dealing specifically with the electrolyte
metabolism. In the pioneer work of Selye, the administration of deoxycorticosterone
14 Renin Angiotensin Aldosterone System and Cardiovascular Disease 209
improved the survival of adrenalectomized rats, but adverse effects (namely

a cardiac necrosis) were also observed [5]. This early observation has trig-
gered, much later, the interest of mineralocorticoid hormones in cardiovascular
diseases.
The identification of aldosterone, a minor hormone of adrenal gland, was diffi-
cult for several reasons, including the fact that plasma glucocorticoids concentra-
tions was much higher than those of aldosterone, that both the hormones and their
receptors have very similar structure, and finally that both types of receptors have
significant, while different, affinities for the two hormones.
14.3 Vascular Effects of Aldosterone

Aldosterone has deleterious effects on both the vascular structure and the function.
Several groups of investigators have observed the induction of a peri-inflammatory
phenotype in the heart of rats treated with high dose of aldosterone + salt-enriched
diet (review in [6]). The increase of the inflammation markers such as Cox-2 and
MCP-1 is seen from the first week on, making the proliferation of inflammatory cells
around the coronary arteries among the first events leading to fibrosis. The causal
role of oxidative stress is suggested by the fact that spironolactone and antioxidants
prevent these changes independently in the coronary [7] or peripheral [8] arteries,
and aldosterone stimulates the expression of the NADPH oxidase in macrophages
[9]. Again, a cooperation between aldosterone and Ang II was found in the release
of free radicals that can lead to a deterioration of arterial smooth muscle cells [10].
The target of the deleterious effects of high concentrations of aldosterone is clearly
the vessels. Nevertheless, it remains to understand the earliest stages. Weber and
his colleagues described an early drop of intracellular magnesium and calcium con-
centrations in monocytes and lymphocytes of rats treated with aldosterone-salt [11].
Several markers of oxidative stress were increased in plasma (alpha-1-antiproteinase
activity) and in heart (gp91phox subunit of NADPH oxidase and 3-nitrotyrosine) of
these animals. If such ionic changes were not observed in cardiac cells, this work
suggests that they may also exist and induce the release of free radicals, coronary
lesions, perivascular, and finally interstitial fibrosis. Finally, in the transgenic mice
model overexpressing the aldosterone synthase within the heart, original effects
have been observed on coronary vasomotricity. In this model characterized by a
moderate increase (1.7 times) of intra-cardiac aldosterone with unchanged plasma
level, the vasodilatory response to acetylcholine is abolished in male transgenic
mice [12]. The mechanism of the damage is the inhibition of the BKCa potas-
sium channels of coronary smooth muscle cells. Interestingly, the cardiac structure
and function remain normal, and the only potentially harmful event discovered to
date is this coronary alteration. In fact, the results of this transgenic study suggests
that a slightly increased concentration of aldosterone (reaching a level observed
in pathological situations) can induce a coronary dysfunction, which is silent in
resting conditions but that may make these animals vulnerable to an increase in
cardiac work.
14.4 Fibrogenic and Arrhythmogenic Effects of Aldosterone

One of the best-documented deleterious effect of aldosterone is cardiac fibrosis,
with adverse consequences on the pump function and an arrhythmogenic effect.
Besides the experimental works, a relationship between mortality and the initial car-
diac fibrosis and a reduced cardiac fibrosis by spironolactone treatment is observed
in a subgroup of patients of the RALES study [13]. Ang II is probably also involved
in the genesis of fibrosis since aldosterone increases cardiac AT1 receptors density
[14], and the expression of angiotensin-converting enzyme, ACE, in rat cardiomy-
ocytes [15]. A pro-arrhythmogenic effect of aldosterone (which might partly depend
on fibrosis) is suggested by several observations. In hypertensive patients, for the
same level of hypertension, atrial fibrillation is much more frequent among those
with primary aldosteronism [16]. In failing rats, spironolactone significantly reduces
fibrosis and atrial ventricular excitability [17]. Transgenic mice overexpressing the
MR in cardiomyocytes have a normal heart function, but show arrhythmia and sud-
den death [18]. Other effects can be evoked, which can also affect the cardiac func-
tion. Finally, Vassort et al. observed an increase in the slow iCaL calcium current
and a decrease of the Ito transitory potassium current in isolated cardiomyocytes,
which could change the electrical characteristics of these cells [19].
14.5 The Signaling Pathways of Aldosterone
As every steroid hormones, aldosterone binds to a cytoplasmic receptor, the MR.

The hormonereceptor complex dimerizes, migrates into the nucleus, and binds to
a specific DNA sequence, which triggers transcription of target genes. In epithelial
cells (kidney, colon, salivary glands, skin, etc.), the induced genes as the amiloride-
sensitive sodium channel ENaC, Na,K-ATPase, and SGK kinase are key factors
in the control of sodium reabsorption. The aldosteroneMR complex binds to the
glucocorticoid responsive element (GRE). The existence of tissue-specific proteins
able to modulate the response of the GRE according to the bound hormone (aldos-
terone or cortisol) has been postulated, but not evidenced to date. In vascular cells,
some target genes are identified, such as the endothelial NO synthase (NOS3) down-
regulated by aldosterone (review in [20]), or the smooth muscle cell BKCa repo-
larizing potassium channel whose coronary expression is reduced by aldosterone,
inducing a decrease in their response to acetylcholine and thus a decrease of the
coronary reserve [21].
Besides this mode of action involving the classical MR, aldosterone induces
cellular responses within minutes that modulate the concentration of intracellular
Ca2+ and cAMP, the activity of the NaH exchanger, and the phosphorylation of
molecules such as the PKC, the EGF receptor (EGFR), and several MAP kinases.
These responses are independent of the MR activation, as suggested by the rapid
onset of the action, and a membrane receptor has been unsuccessfully sought.
But there is also a third mode of action of aldosterone, activated by binding of
aldosterone on the MR and triggering in some minutes (i.e., without synthesis of
proteins) the activation of the EGFR signaling pathway together with an increased
phosphorylation of ERK1/2 and JNK 4 kinases. All these mechanisms are associ-
ated with the inflammation and vascular remodeling leading to fibrosis.
14.6 The Receptors of Corticosteroid Hormones

The MR and GR belong to the nuclear hormone receptors superfamily, and they
have a pronounced sequence homology. The two receptors bind glucocorticoids
(cortisol in humans and corticosterone in rats and mice) with a strong affinity. How-
ever, aldosterone binds to MR with a strong affinity, while its affinity for the GR
is much lower. Because plasma aldosterone levels are 3 orders of magnitude lower
than those of cortisol and corticosterone, glucocorticoids should occupy most of the
MRs. However, this theoretical excess is decreased by a 10 factor by the impor-
tant binding of glucocorticoids to plasma transcortin (only 3% of cortisol is free in
plasma), while the binding of aldosterone to albumin is lower (30% of plasma aldos-
terone is free). In addition, transfection studies have shown that cortisol has a trans-
activation activity of MR 10 times lower than that of aldosterone despite identical
binding affinities. In addition, the cortisolMR complex is less stable than the com-
plex aldosteroneMR, because there are differences in the conformation changes of
MR induced by the hormone binding. This leads to a 24 time faster dissociation
of the cortisolMR complex than that of the aldosteroneMR complex. Finally, the
exact mechanism of entry of cortisol and aldosterone in the cell is not clear, and
there might be other differences between these steroids due to the aldosterone 11-18
hemi-acetal group. A first conclusion is that aldosterone seems disadvantaged com-
pared to cortisol to bind to MR, but probably not as much as the ratio of plasma
concentrations suggests. But in these conditions, how does aldosterone have a spe-
cific action?
The answer depends on the cell type. In epithelial cells and in endothelial and
smooth muscle cells, which express the MR, the binding of aldosterone on the MR
is made possible by the presence of the enzyme 11-HSD2 (11-beta-hydroxysteroid
dehydrogenase type II), which metabolizes cortisol and corticosterone in their inac-
tive cortisone and 11-dehydro-corticosterone metabolites. In contrast, in cells such
as cardiomyocytes which express the MR but not the 11-HSD2, the MR is proba-
bly mostly occupied by glucocorticoids. In this view, aldosterone can probably have
no significant MR-dependent action. But, as outlined above, there are possible other
mechanisms that allow the binding of aldosterone on the MR, even in the absence of
11-HSD2. The studies on isolated cardiomyocytes evidence effects of aldosterone
on calcium or potassium currents, but these effects are observed using high concen-
trations of aldosterone in a milieu containing few or no glucocorticoids.
The situation is different for the cortisolmineralocorticoid receptor
complex. Under physiological conditions, this complex is inactive, but under
pathophysiological conditions it may be activated and function like the aldosterone
mineralocorticoid receptor complex [22]. It would be interesting to compare the
effects of aldosterone and those of glucocorticoids in the presence of a GR inhibitor
in order to identify the MR-dependent effect alone on isolated cardiomyocytes. One

might think they are identical, since both hormones are able to link the MR with
the same affinity, and indeed corticosterone activates the MR in smooth muscle
cells and triggers rapid responses of MAP kinase and ERK1/2 pathways, which can
have adverse consequences on the vessel [23]. To complicate the matter, several
studies have shown that cortisol may block the action of aldosterone, suggesting
that in many cases cortisol binds the MR and acts as a MR antagonist. Transgenic
mice overexpressing the MR in cardiomyocytes [18] or in other cell types are a
powerful and elegant means to explore these mechanisms. It is therefore important
to realize that MR antagonists inhibit the effetcs of aldosterone in cells containing
the 11-HSD2, but may also inhibit the action of cortisol in cells which do not
express this enzyme. In the case of the few specific MR antagonist spironolactone,
the actions mediated by the GR may also be partially inhibited.
14.7 Interferences Between Aldosterone and Angiotensin II

One of the difficulties in interpreting the effects of aldosterone depends on the inter-
actions between the signaling pathways activated by other hormones or receptors,
namely Ang II and MR (review in [24]). Several laboratories have demonstrated
that aldosterone stimulates the transcription of the AT1 receptor (AT1R) of Ang II,
and ACE, which results in an increased local production of Ang II. On the other
hand, the Ang II-dependent increase in collagens is at least in part an aldosterone-
dependent effect [25]. It has been recently shown in hamster-isolated cardiomy-
ocytes that eplerenone inhibits the intracrine action of Ang II on inward calcium
current and reduces drastically the effect of extracellular Ang II on the ICa current
[26]. Since aldosterone reverse eplerenone effetcs, these results show that the MR
is an essential component of the intracrine renin angiotensin aldosterone system.
Interestingly, the proliferation of vascular smooth muscle cells is stimulated by a
combination of low doses of aldosterone and of Ang II, while aldosterone or Ang
II alone have no effects [27]. Similarly, aldosterone increases neovascularization
in an in vivo model of ischemia secondary to right femoral artery ligature in mice
[28]. Inhibition of these effects by valsartan shows that the pro-angiogenic action
of aldosterone involves the AT1R. Finally, Ang II can directly activate the MR in
the coronary and aortic CML [29]. So there are interactions between the effects of
aldosterone and those of Ang II, reinforcing the therapeutic interest of combining
MR and AT1R inhibitors in cardiovascular diseases.
14.8 Conclusion
There are still many avenues to explore. In addition to the mechanisms, it is perti-
nent to determine whether the increase in aldosterone in common diseases such as
diabetes, hypertension, and left ventricular hypertrophy is an additional risk factor.
For example, in metabolic syndrome plasma aldosterone is increased [30]. In rat

cardiomyocytes, the local production of aldosterone modulates potassium currents
and increases oxidative stress, but only in male diabetic animals [31]. Preliminary
results from our laboratory show that in the mouse heart, a slight increase in aldos-
terone exerts a protective role against the deleterious effects of type 1 diabetes. This
is another example of the complexity of the effects of aldosterone which appear to
vary depending on the concentration of the hormone, of gender, and of the cellular
environment.
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Chapter 15
Renin Angiotensin System and Atherosclerosis
Changping Hu and Jawahar L. Mehta
Abstract Renin angiotensin system (RAS) regulates a host of biological func-

tions in the body, including maintenance of vascular tone. All components of RAS
have been identified in atherosclerotic tissues and are believed to regulate oxidation
of LDL-cholesterol, endothelial function, formation of foam cells, smooth muscle
cell proliferation, and collagen deposition that covers the atherosclerotic plaque. In
keeping with the concept of the pathogenic role of RAS activation in atherosclero-
sis, inhibition of renin formation, angiotensin-converting enzyme and angiotensin
II type 1 receptor activation, all have been shown to inhibit atherogenesis, primar-
ily via the inhibition of oxidative stress. Recent studies from our laboratory show
that delivery of angiotensin II type 2 receptor cDNA with adeno-associated virus
as vector can inhibit the process of atherogenesis in the LDL receptor-knockout
mice. This is associated with a marked increase in the expression of endothelial
constitutive nitric oxide synthase, heme-oxygenase-1, and Akt activation and a dra-
matic reduction in the expression of LOX-1 and activity of NADPH oxidase and the
redox-sensitive transcription factor NF-kB as well as the pro-inflammatory activity
of p38 component of MAP kinase. This new information has the potential to lead to
the development of novel therapeutic strategies directed at different components of
RAS either in combination or as stand-alone therapy.
Keywords Angiotensin II Atherogenesis LDL receptor-knockout mice LOX-1
15.1 A Brief Overview of Renin Angiotensin System
As recognized by all [1], renin is a 40,000-dalton glycoprotein that is expressed,

stored, and released in a regulated manner by the juxtaglomerular cells of the
kidneys. Renin, initially synthesized as an inactive zymogen known as pro-renin,
C. Hu (B)
Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences and the Central
Arkansas Veterans Healthcare System, Little Rock, AR, USA
e-mail: huchangping@yahoo.com

216 C. Hu and J.L. Mehta
has exquisite substrate specificity, and its only known substrate is angiotensino-
gen. Angiotensinogen, mainly synthesized in the liver, is the only precursor of
angiotensin peptides. Renin cleaves the N-terminus of circulating angiotensinogen
to form angiotensin (Ang) I, which is an inactive peptide for which no specific
receptor has so far been established. Ang I, in turn, is cleaved by the endothe-
lial cell-associated or soluble dipeptidyl carboxypeptidase angiotensin-converting
enzyme (ACE) to form the biologically active Ang II.
Among many receptors that Ang II acts on, the two main receptors relevant to
cardiovascular system are type 1 (AT1) and type 2 (AT2); both stem from single
genes found on chromosome 3 and X, respectively. Both AT1 and AT2 receptors
are seven-transmembrane, G-protein-coupled receptors. Both are expressed endoge-
nously at very low levels. The AT1 receptors are mainly expressed in blood vessels,
adrenal cortex, liver, and brain. The AT2 receptors are found primarily in fetal tis-
sues, adrenal medulla, and uterus. While AT2 receptors are present in large numbers
at birth, their number declines rapidly so that in the adult the AT1 receptors predomi-
nate [2, 3]. Most of the known physiologic functions of Ang II appear to be mediated
by the activation of AT1 receptors, whereas the AT2 receptor activation appears to
mediate apoptosis and growth inhibition as well as fetal growth regulation [4, 5].
Activation of AT1 receptors by Ang II stimulates a variety of intracellular sig-
nal pathways, including those typically activated by G-protein-coupled receptors,
growth factor receptors, and cytokines, as well as events leading to the regulation
of receptor function, such as phosphorylation and internalization of the receptor
[6]. AT1 receptor activation also regulates gene expression, leading to a variety of
growth-related responses, including activation of receptor and non-receptor protein
tyrosine kinases [1, 2]. In addition, AT1 receptor activation by Ang II stimulates
signal transducers and activators of transcription pathway, small G proteins, and
expression of other important regulatory enzymes, such as phospholipase D, phos-
pholipase A2, and NAD(P)H oxidase [1, 2]. Ang II binding to AT1 receptors also
stimulates the internalization and processing of the ligandreceptor complex [1, 2].
Ang II exerts most of the physiologic functions of the renin angiotensin system
by activating membrane-bound receptors, whereas angiotensinogen and Ang I are
inactive peptides for which no function has been described so far [1, 2].
15.2 Renin Angiotensin System, Oxidative Stress, and LOX-1

Expression
Ang II activates NADPH oxidase system, perhaps the most powerful system in
mammalian species, resulting in the generation of reactive oxygen species (ROS),
including superoxide anions and hydrogen peroxide [7]. Many of the effects of Ang
II are thought to be related to the release of ROS. When the effect of ROS cannot
be adequately countered by innate antioxidant system, this state is often termed as
oxidative stress [8]. Oxidative stress is present in a variety of cardiovascular disease
states, such as atherosclerosis, hypertension, and myocardial ischemia [8]. While
some amount of oxidative stress is necessary for survival, release of large amounts
15 Renin Angiotensin System and Atherosclerosis 217
of oxidants is clearly pathologic. Studies from our and others laboratories have
shown that Ang II via redox-sensitive NF-B pathway leads to the expression of a
lectin-like oxidized low-density lipoprotein (LDL) receptor (now commonly known
as LOX-1) in endothelial cells as well as smooth muscle cells [9]. LOX-1 activa-
tion by oxidized LDL-cholesterol (ox-LDL) also via the activation of NF-B and
Oct-1 pathways leads to the transcription of AT1 receptors [10, 11]. Thus, there is a
positive feedback loop between Ang II and dyslipidemia involving redox-sensitive
intracellular pathways [12]. This aspect of renin angiotensin system biology and its
relevance will be discussed in detail later in this chapter.
15.2.1 Oxidative Stress in Atherosclerosis
Atherosclerosis is the most common aging-associated disease in the developed

countries, and its incidence is rapidly rising in the developing countries. All
atherosclerosis risk factors, such as diabetes, hypertension, dyslipidemia, and smok-
ing are associated with excessive production of ROS well beyond the capability of
endogenous antioxidants to scavenge them [13]. Increased oxidative stress plays an
important role in various steps in atherosclerosis as exemplified by the following
[12, 14]: (i) oxidative stress induces the oxidation of LDL-cholesterol, resulting in
the formation of ox-LDL and its uptake by vascular wall components in large part
by activation of LOX-1 and other scavenger receptors; (ii) oxidative stress activates
endothelial cells, resulting in the expression of adhesion molecules which lead to
the recruitment and adhesion of monocytes to the endothelium and their subsequent
migration into the subendothelial space; (iii) oxidative stress is responsible for the
release of metalloproteinases (MMPs) which cause the disruption of atherosclerotic
plaque; (iv) oxidative stress causes the proliferation and migration of smooth mus-
cle cells and fibroblasts, leading to the thickening of vessel wall and narrowing of
the lumen; and (v) oxidative stress activates platelets and stimulates the formation
of the occlusive thrombus in the narrowed arterial lumen.
Both ox-LDL and Ang II facilitate the initiation and propagation of oxidative
stress in the blood vessel wall [1517]. Previous in vitro studies indicated that treat-
ment directing at ox-LDL or renin angiotensin system have antioxidant effects in
vascular wall components [18, 19]. Further, therapies targeted at hyperlipidemia
(such as with statins) or renin angiotensin system blockers, especially AT1 receptor
blockers, have potent antioxidant and antiatherosclerotic effects in laboratory ani-
mals and humans [20, 21]. Recent evidence supports the concept that ox-LDL and
Ang II interact synergistically in the context of oxidative stress [22]. This idea is
based on the following considerations: first, there are striking similarities between
the effects of ox-LDL and Ang II, especially those related to oxidative stress, in vas-
cular endothelial cells and smooth muscle cells as well as in animal tissues [1517];
second, the blockade of AT1 receptors normalizes the activity of NADPH oxidases
and reduces atherosclerotic plaque burden in animals fed a high-cholesterol diet
[23]; and third, ox-LDL and Ang II together induce cumulative injurious effects
in cells, and importantly, antioxidants such as vitamin E attenuate these injurious
effects [24].
15.2.2 Role of LOX-1 in Atherosclerosis

LOX-1 is a 52-kD surface receptor initially identified in bovine aortic endothelial
cells [25]. This receptor, upregulated by ox-LDL, is responsible for binding and
uptake of ox-LDL in endothelial cells [26, 27]. The contributory role of LOX-1 in
atherogenesis is supported by several lines of evidence [14]: (i) LOX-1 binds, inter-
nalizes, and degrades ox-LDL in endothelial cells; (ii) ox-LDL-induced endothelial
dysfunction is mediated by LOX-1 activation; (iii) besides ox-LDL, other media-
tors of atherosclerosis, such as angiotensin II, cytokine TNF-, sheer stress, ROS,
and advanced glycation end-products, upregulate LOX-1; (iv) LOX-1 is dynami-
cally upregulated by pro-atherogenic conditions, such as diabetes, hypertension, and
dyslipidemia; (v) LOX-1 is present in atheroma-derived cells and in human and ani-
mal atherosclerotic lesions; (vi) LOX-1 blockade modulates endothelial function in
coronary arterioles in atherosclerotic ApoE knockout mice [28] as well as in LDL-
receptor knockout mice [29]; and most importantly LOX-1 deletion in the LDLR
knockout mice significantly reduced atherogenesis [29].
LOX-1 is intimately involved in the generation of ROS in inflammatory cells
and endothelial cells [30, 31], and in a positive feedback fashion ROS directly
induces LOX-1 expression. LOX-1 has also recently been identified on the surface
of platelets [32], which plays an important role in platelet aggregation and thrombus
formation [33].
In recent studies, we established that fibroblasts proliferate and express colla-
gen signal rapidly in response to Ang II [16]. Chen and colleagues [9] in our
laboratory observed that NF-B activation plays a critical role in Ang II-induced
LOX-1 promoter activation. As is well known, vascular fibrosis is stimulated
by the cytokine transforming growth factor 1 [34]. Hu et al. [35] observed
that the transforming growth factor 1-mediated increase in collagen synthe-
sis is markedly attenuated in fibroblasts from the LOX-1 knockout mouse, sug-
gesting that LOX-1 is an important link in collagen formation in fibroblasts
in response to growth factors such as angiotensin II and transforming growth
factor 1.
Ishigaki et al. [36] reported recently that LOX-1 expressed ectopically in the liver
with adenovirus administration in apoE-deficient mice, another animal model of
hypercholesterolemia and atherosclerosis, removed ox-LDL from circulating blood
and possibly decreased systemic oxidative stress, resulting in the prevention of
atherosclerosis. In keeping with this concept, LOX-1 transgenic mice display accel-
erated intramyocardial vasculopathy and a marked increase in atheroma-like lesion
areas [37].
15.2.3 Cross-Talk Between Oxidized-LDL and Renin Angiotensin

System
Ox-LDL has been shown to increase the expression of ACE and AT1 receptors
in cultured endothelial cells and smooth muscle cells [10, 38]. Atherosclerotic
lesions in hyperlipidemic animals show activation of renin angiotensin system
[39]. Studies in human atherosclerotic tissues have also confirmed the activation
of renin angiotensin system, particularly in the regions prone to plaque rupture
[40]. Conversely, there is evidence for downregulation of AT1 receptor expres-
sion with lipid-lowering reagents, particularly 3-hydroxy-3-methylglutaryl coen-
zyme A reductase inhibitors (also known as statins), in vascular smooth muscle cells
and endothelial cells [38, 41], and in hyperlipidemic atherosclerotic animals and
humans [41, 42].
It also should be noted that while high levels of LDL-cholesterol upregulate
various components of renin angiotensin system, Ang II via AT1 receptor activa-
tion facilitates cellular cholesterol biosynthesis, oxidation of LDL, and uptake of
ox-LDL by vessel wall components [12].
15.2.4 Aneurysm Formation in Atherosclerosis Role of Lipids

and Renin Angiotensin System
Aneurysm is defined as a permanent dilation of the arterial wall, which is char-

acterized by outward vascular remodeling, both in vascular dimension and in
structure. Processes such as proteolysis, inflammation, and ROS formation are
important during aneurysm formation. As discussed earlier, ACE and downstream
regulators such as Ang II and AT1 receptor activation are also involved in this
process [43].
ACE is expressed in the aneurysmal vascular wall, both in human tissues
and in animal models. In aneurysmal aortic specimens obtained during opera-
tive repair in patients, ACE activity was found to be significantly increased com-
pared to that in the normal segments [43]. ACE-positive cells, determined by
immunohistochemical staining, were mainly macrophages, both in the media and
in the intima [43]. Chymase activity was also significantly increased in these
specimens and chymase-positive cells were mainly mast cells in the media and
adventitia [43]. In a rabbit model for aneurysm formation, by elastase perfusion,
ACE protein levels in the aortic wall increased during aneurysm growth over
time [43].
In mice, infusion of Ang II induced aneurysm formation independent of changes
in blood pressure. This was shown in a hyperlipidemic setting in ApoE knockout
mice as well as in the wild-type C57BL6 mice, although aneurysm formation was
smaller in the wild-type mice [43]. This implies a critical role of hyperlipidemia
in the development of aneurysms in response to Ang II. In this hyperlipidemic
mice model, proteolytic processes are clearly involved since the broad-spectrum
matrix MMP inhibitor doxycycline reduced the severity of aneurysm formation [43].
Ang II-induced aneurysm also displays characteristic inflammatory features, which
fits with the role of Ang II in inflammatory processes [43]. These studies suggest
that Ang II, partly because of its inflammatory effects, is an important initiator of
aneurysm formation.
15.2.5 Studies of Angiotensin-Converting Enzyme Inhibitors and

Angiotensin Type 1 Receptor Antagonists in Atherosclerosis
ACE inhibitors act by inhibiting the conversion of Ang I to Ang II. In addition, ACE
inhibitors decrease the breakdown of bradykinin. Furthermore, ACE inhibitors,
which increase tissue and plasma levels of angiotensin (17), may also improve
the fibrinolytic balance by decreasing plasminogen activator inhibitor-1 formation
via Ang IV and/or reduce Ang II-induced activation of angiotensin type 4 receptors
[44]. Since tissue ACE is highly expressed in human atherosclerotic plaques [45],
where it is localized in areas of clustered macrophages, and is significantly increased
in patients with unstable angina [44], it has been suggested that blocking ACE will
prevent plaque fissuring, thrombosis, and rupture.
A direct anti-atherogenic effect of ACE inhibitors has been shown in a variety
of animal models. A large body of data on the beneficial effects of ACE inhibitors
comes from landmark clinical trials such as the Heart Outcomes Prevention Eval-
uation (HOPE) study and the EUropean trial of Reduction Of cardiac events with
Perindopril in stable coronary Artery disease (EUROPA), which included patients
with documented coronary artery disease (CAD), risk factors, and preserved left
ventricular function. These two, and several other, trials demonstrated convincingly
that cardiovascular events could be reduced in patients who were treated with an
ACE inhibitor [46, 47]. These large morbidity and mortality trials clearly support
the role of ACE inhibitors in the treatment of atherosclerotic vascular diseases.
The AT1 antagonists bind to the AT1 receptor with high affinity. These agents
reduce the activation of AT1 receptor-mediated actions of Ang II more effec-
tively than ACE inhibitors since the latter do not reduce alternative, non-ACE Ang
II-generating pathways, such as those involving chymase, or cathepsin G [44].
In contrast to ACE inhibitors, AT1 antagonists indirectly activate AT2 receptors.
The importance of AT2-mediated effects is not clearly established. Nevertheless,
recent studies suggest that AT2 receptors exert anti-proliferative, pro-apoptotic, and
vasodilatory actions, and may have a modest effect on promoting bradykinin release
[44]. ACE inhibitors increase angiotensin (17) levels more than AT1 antagonists,
and this may result in additional beneficial cardiac and vascular effects. Moreover,
ACE inhibitors increase the levels of a number of other ACE substrates that are not
angiotensin peptides, including bradykinin. The increase in bradykinin levels may
also contribute to the beneficial cardiovascular effects of ACE inhibitors. Whether
or not these distinct pharmacological differences between AT1 antagonists and ACE
inhibitors result in significant differences in therapeutic outcomes is not known at
present.
Experimental studies in mice, rabbits, and primates have demonstrated convinc-
ingly that AT1 receptor antagonists decrease cardiac and arterial medial hypertrophy
and reduce the development of atherosclerotic lesions [44]. A number of studies
have demonstrated that AT1 antagonists, especially losartan, may exert additional
anti-aggregatory and anti-inflammatory actions [44]. These effects may be of poten-
tial interest especially with regard to the treatment of patients with acute manifesta-
tions of atherosclerotic disease such as acute myocardial infarction.
Clinical trials have shown that AT1 receptor antagonists can effectively lower
blood pressure and may positively influence atherosclerosis in hypertensive patients.
However, unfortunately, there are no randomized, clinical trials to date on the effects
of AT1 receptor antagonists on the anatomic progression of atherosclerotic vascular
disease. The Losartan Intervention for Endpoint reduction (LIFE) in hypertension
study showed lower primary event rate in the losartan-based treatment group. Inter-
estingly, most benefit was related to a 25% reduction in the rate of strokes [44].
A renoprotective effect of AT1 receptor antagonists was convincingly demon-
strated in the Irbesartan Diabetic Nephropathy Trial (IDNT) [48], the Reduc-
tion of Endpoints in NIDDM (NonInsulin-Dependent Diabetes Mellitus) with
the Angiotensin II Antagonist Losartan (RENAAL) study [48], and the IRbe-
sartan MicroAlbuminuria type 2 diabetes mellitus in hypertensive patients trial
(IRMA 2) [44]. Cardiovascular end-points were pre-specified secondary outcomes
in the RENAAL and IDNT studies. In the RENAAL study, the rates of fatal and
non-fatal cardiovascular events did not differ significantly between the study groups,
with the exception of hospitalization for heart failure, for which the risk was reduced
by 32% in the losartan group. In the IDNT study, there was no significant dif-
ference in cardiovascular outcome between study groups. However, these studies,
which enrolled relatively small number of patients, were statistically underpowered
to show significant differences in cardiovascular outcome. It remains speculative
whether larger trials may show more clear-cut cardiovascular benefits and whether
the renoprotective benefits demonstrated may be at least in part mediated by vascu-
lar protective actions of AT1 receptor antagonists.
To date, there is only small body of data from large-scale randomized trials
with AT1 receptor antagonist in chronic atherosclerotic vascular diseases. Further,
whether AT1 receptor antagonists are superior to ACE inhibitors has not been clearly
established.
15.2.6 Studies with Renin Inhibitors in Atherosclerosis
Renin is the rate-limiting enzyme in the production of all angiotensin peptides. It

has been shown that in fat-fed LDL receptor-deficient mice administered the novel
renin inhibitor aliskiren over a broad dose range, renin inhibition results in striking
reductions of atherosclerotic lesion size in both the aortic arch and the root [49].
Subsequent studies demonstrated that cultured macrophages express all compo-
nents of the renin angiotensin system. To determine the role of macrophage-derived
angiotensin in the development of atherosclerosis, renin-deficient bone marrow was
transplanted to irradiated LDL-receptor deficient mice and a profound decrease in
the size of atherosclerotic lesions was observed. In similar experiments, transplan-
tation of bone marrow deficient for Ang II type 1a receptors failed to influence
lesion development. These findings suggest that renin-dependent angiotensin pro-
duction in macrophages does not act in an autocrine/paracrine manner. Furthermore,
in vitro studies demonstrated that co-culture with renin-expressing macrophages
augmented monocyte adhesion to endothelial cells. Therefore, although previous
work suggests that angiotensin peptides have conflicting effects, renin inhibition
profoundly decreases lesion atherosclerotic lesion development in mice.
15.2.7 Reduction in Atherosclerosis with Combination of Statins

and Angiotensin-Converting Enzyme Inhibitors/AT1
Receptor Antagonists
The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) induce
regression of atherosclerosis and reduce cardiovascular-related morbidity and mor-
tality in patients with and without coronary artery disease [50]. Data from both
primary and secondary prevention trials demonstrate that statins reduce the risk of
cardiovascular events well beyond their hyperlipidemic effect. Recent evidence sug-
gests that statins and blockers of the renin angiotensin system may share the ability
to interfere with a number of key atherogenic processes, including smooth muscle
cell migration/proliferation, inflammatory reactions, platelet adhesion/aggregation,
macrophage activation, and mediator expression [50]. Statins reduce Ang II-induced
inflammation within atherosclerotic plaques, leading to stabilization of these lesions
and also to downregulation of AT1 receptor expression in isolated vascular smooth
muscle cells. Statin-mediated reduction of AT1 receptor gene expression may impair
the effects of Ang II on intracellular signaling, which may ultimately lead to dimin-
ished activation of NADPH oxidase. In studies by Chen et al. [51], combination of
a potent statin rosuvastatin and an AT1 receptor blocker candesartan almost com-
pletely blocked atherogenesis in ApoE knockout mice. Further, there was a dramatic
cumulative effect of these agents on markers of oxidative stress, inflammation, and
expression of MMPs. These effects may well have been a direct effect of total block-
ade of LOX-1 expression.
Hyperlipidemia is a frequent component of the metabolic syndrome, and
pre-clinical studies show that statins in combination with ACE inhibitors or AT1
receptor antagonists may facilitate blood pressure control via a mechanism inde-
pendent of lowering of plasma lipids. Nazzaro and colleagues [52] examined the
individual and combined effects of ACE inhibitors and statins on blood pressure in
patients with coexisting hypertension and hypercholesterolemia. Monotherapy with
enalapril or simvastatin resulted in a reduction in blood pressure, but administra-
tion of both agents produced greater blood pressure reduction than with either agent
alone. The propensity of lipid-lowering agents to provide additive benefit to ACE
inhibitors in lowering blood pressure has also been noted in several other studies.
Collectively, these studies provide strong evidence of a close interplay between
dyslipidemia and the renin angiotensin system. Investigation of combined effects of
statins and ACE inhibitors or AT1 receptor antagonists on management of patients
with resistant hypertension is of considerable interest, because endothelial dys-
function may reduce the antihypertensive action of drugs that predominantly act
via restoration of vascular endothelial function. The combination of these drugs
may provide an improved approach to the management of endothelial dysfunction,
inflammation, oxidative stress, and atherosclerosis.
15.3 Angiotensin II Type 2 Receptor Upregulation in

Atherogenesis
There is substantial evidence that Ang II mediates atherosclerosis development

mainly via the activation of AT1 receptors [1, 44]. Moreover, deletion of the AT1a
receptor in ApoE knockout mice prevents atherosclerosis formation, underlining the
importance of this receptor subtype in this pathology [53]. Since there is growing
evidence that the activation of the AT2 receptor may act opposite to the activation
of the AT1 receptor, the role of AT2 receptor in atherogenesis has been examined.
Iwai et al. [54] showed that deletion of AT2 receptor exaggerated atherosclerosis
in Apo-E deficiency mice. Others, however, reported that AT2 receptor deficiency
had no effect on atherogenesis in LDL receptor deficient [55] and ApoE deficient
mice [56]; yet AT2 receptor deficiency augmented the cellularity of atherosclerotic
lesions. Johnasson et al. [57] found that administration of an AT2 receptor antago-
nist had no effect on Ang II-accelerated atherosclerosis in Apo-E deficiency mice.
Results presented in these studies are discrepant, and, therefore, no definitive con-
clusion concerning the role of AT2 receptors in atherogenesis can yet be proposed.
The reasons advocated for this discrepancy can be linked to sex, strain of mice, and
the model of Ang II-induced atherogenesis. In fact, in all these studies, increased
circulating Ang II levels were achieved by infusion of high doses of exogenous Ang
II. Using this model, the physiologic regulatory mechanisms of Ang II secretion
were not taken into account. In addition, it has been recently shown that wild-type
mice treated with an AT1 receptor antagonist have decreased tissue Ang II, despite
increased plasma levels of Ang II [1]. This suggests that blockade of the AT1 recep-
tor rather than enhanced stimulation of AT2 receptors may account for many of the
beneficial effects seen with AT1 receptor antagonists. Moreover, in clinical trials
and in everyday clinical practice, no substantial difference in the beneficial effects
has been seen with either ACE inhibitors or AT1 receptor antagonists, suggesting
that the precise contribution of AT2 receptors still remains to be established.
15.3.1 Reduction in Atherosclerosis with Gene Therapy Directed at

AT2 Receptor Overexpression
As mentioned above, AT2 receptor deletion was shown to exaggerate atheroscle-

rosis in ApoE-deficient mice [54]. Therefore, we hypothesized that AT2R
(agtr2) overexpression might inhibit atherogenesis [58]. We prepared recombinant
adeno-associated virus type-2 (AAV) carrying AT2 receptor cDNA (AAV/AT2R)
(Fig. 15.1), and homozygous LDL receptor-deficient mice were given AAV/AT2R,
AAV/Neo or saline. All mice were placed on a high-cholesterol diet. After 18
weeks, AT2 receptor was found to be overexpressed systemically in AAV/AT2R-
treated mice. Atherogenesis in aorta was reduced in the AAV/AT2R group by 50%
compared to other LDLR knockout mice given saline of AAV/Neo. Expres-
sion of NADPH oxidase, nitrotyrosine (a marker of oxidative stress), and the
Fig. 15.1 Construction of AAV/AT2R vector and generation of rAAV stocks. Mouse AT2R (agtr2)
cDNA was generated, sequenced, and ligated into an AAV vector, dl6-95. Recombinant AAV vec-
tor is referred to as AAV/AT2R. The titer of purified virus, in encapsidated genomes per milliliter
(eg/ml), was calculated by dot-blot hybridization and determined to be about 1011 eg/ml
redox-sensitive transcription factor NF-B was increased in aortic tissues of the

LDL receptor-deficient mice given saline or AAV/Neo, but not in mice with AT2
receptor upregulation. Expression of endothelial nitric oxide synthase and heme-
oxygenase-1 was decreased and that of LOX-1 increased in the LDL receptor-
deficient mice, but not in the mice with AT2 receptor overexpression. Further, Akt-1
phosphorylation was reduced in the LDL receptor-deficient mice, but not in the
mice with AT receptor 2 overexpression (Fig. 15.2). Thus, AT2 receptor upregu-
lation can reduce atherogenesis, possibly by modulating oxidative stress and the
pro-inflammatory cascade, mediated via Akt-1, and may be an important thera-
peutic approach in atherosclerosis. However, these data need to be replicated in
other experimental animals. If this approach proves successful, use of strategies
designed at overexpression of AT2 receptors alone or with AT1 receptor antagonist
therapy might be a useful approach in the treatment of atherosclerosis and related
disorders.
15.3.2 Reduction in Collagen Deposition with AT2 Receptor

Upregulation
We also found that LDL receptor-deficient mice treated with saline or AAV/Neo
exhibited extensive collagen accumulation in the aortic wall, which was reduced
Fig. 15.2 (A) Representative examples of reduction in atherogenesis in LDLR-deficient mice

given AAV/AT2R. The administration of AAV/Neo had no effect on the extent of atherosclerosis.
(B) The administration of AAV/AT2R altered the expression of proteins assisted with atherogene-
sis, such as LOX-1, nitrotyrosine, eNOS, phos-Akt, and HO-1. This alteration most likely occurred
as a result of reduction in NADPH oxidases (p47phox and p22phox ) and activation of NF-B
by about 50% with AT2 receptor overexpression [59]. Further, AT2 receptor
upregulation completely blocked the alterations in the expression of procollagen-
I, osteopontin, fibronectin, CD68, and MMPs (MMP-2 and MMP-9), as well as
phosphorylation of p38 and p44/42 MAPKs. Activity of superoxide dismutase was
reduced in the LDL receptor-deficient mice and it increased with AT2 receptor
upregulation (Fig. 15.3). This study for the first time showed that AT2 receptor
overexpression reduces enhanced collagen accumulation and MMP expression and
activity in atherosclerotic regions via inhibition of pro-oxidant signals. Whether the
reduction in collagen reflects merely a reduction in atherosclerosis or if it would
soften the plaque leading to complications such as plaque rupture cannot be dis-
cerned from the present study.
15.3.3 Mechanisms of the Efficacy of AT2R Upregulation

Enhanced expression of collagen appears to be an inherent part of the atheroscle-
rotic process. Ang II is present in the atherosclerotic regions and activates NADPH
oxidase system. The intense oxidant stress in the atherosclerotic regions stimu-
lates mitogen-activated protein kinases and the redox-sensitive transcription fac-
tors, such as NF-B, followed by upregulation of genes, such as fibronectin,
osteopontin, collagens, and MMPs, which result in the formation of collagen.
Interestingly, excessive collagen deposition is associated with enhanced release
of MMPs. While ox-LDL and Ang II-stimulated LOX-1 activation enhances
oxidative stress and inflammation, and oxidative stress per se upregulates LOX-
1 expression. This process may self-amplify leading to intense collagen depo-
sition in atherosclerotic regions over time. These events are summarized in
Fig. 15.4.
Fig. 15.3 (A) Representative examples of reduction in collagen deposition in atherosclerotic

regions in LDLR-deficient mice given AAV/AT2R. Collagen was estimated by tissue stain-
ing with Trichrome and Picro-serius red and the results were consistent. The administration of
AAV/Neo had no effect on collagen deposition in atherosclerotic regions. (B) The administration of
AAV/AT2R altered the expression of proteins assisted with atherogenesis, such as pro-collagen-1,
osteopontin, fibronectin, extracellular superoxide dismutase (ecSOD), metalloproteinases (MMP-9
and MMP-2), and the inflammatory marker CD68. This alteration most likely occurred as a result
of reduction in NADPH oxidases (p47phox and p22phox ) shown in Fig. 15.2 and activation of phos-
p38MAPK
15.4 Summary, Questions, Future Perspective, and Clinical

Implications
There is accumulating clinical and experimental evidence that the pathways by

which hypertension and dyslipidemia lead to vascular changes may overlap and
that Ang II is involved in restructuring (or remodeling) of the arterial wall in both
atherosclerosis and hypertension. Activation of the renin angiotensin system seems
to be a central pathophysiologic event in the progression of endothelial cell dys-
function to atherosclerosis and subsequent development of clinical syndromes. The
pivotal role of the renin angiotensin system in atherogenesis is highlighted by stud-
ies in animal models as well as in humans indicating that inhibition of the renin
angiotensin system with ACE inhibitors or AT1 receptor antagonists retards the
development of atherosclerotic lesions. In light of a causal and central role of Ang II
in atherogenesis, blockade of the renin angiotensin system has come to represent an
important therapeutic consideration in the prevention and treatment of atheroscle-
rotic disease.
Fig. 15.4 Hypothesized pathways of collagen and MMPs changes modulated by LOX-1 activation
(by angiotensin II) and the inhibition of the cascade by AT2R upregulation
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Chapter 16
Renin Angiotensin System and Aging
Len F. Ferder
Abstract We advance herein the hypothesis that aging and its consequences,
including cardiac hypertrophy, result from actions of Angiotensin II (AII). Exper-
imental findings indicate that hypertension and aging have similar effects on the
structure and function of blood vessels, the heart, and, presumably, the kidney.
Keywords Mitochondria ROS Aging Angiotensin II (AII) AII blockade
AII has been proposed to be a mediator of oxidative stress through the induction
of reactive oxygen species (ROS) in aging and in normal animals.
Mitochondria are a major source of ROS in aging. AII blockade decreases ROS at
the mitochondrial level, thus protecting against both age-related mitochondrial dys-
function and ultrastructural alterations, underscoring the role of renin angiotensin
system (RAS) in the aging process.
Diminished density of AII receptors leading to enhanced sensitivity to AII could
explain partly the development of the aging process.
This hypothesis is supported by our findings that ACE inhibitors and AII receptor
antagonists decrease tissue oxidative state and improve mitochondrial number and
function, leading to higher animal survival and prolonged lifespan.
Organ malfunction is an eventual result of the natural process of aging, which all
species undergo. Modifications in organ structure directly related to aging cause in
humans functional deterioration of varying degrees by, among other things, leading
to replacement of functional parenchyma by fibro-connective tissues and induction
of arteriosclerosis in the blood vessels of several organs. Changes in kidney func-
tion, including its role as an endocrine organ, related to age have been recognized
since the seventies. These changes include alterations in the regulation of the renin
angiotensin system (decreased renin production and secretion) and in the endocrine
and paracrine functions of their product angiotensin II (AII), both of which are
L.F. Ferder (B)

Departments of Physiology, Pharmacology and Medicine, Ponce School of Medicine, Ponce, PR
e-mail: leferder@psm.edu

232 L.F. Ferder
dependent on angiotensin II receptors [1, 2]. We advance herein the hypothesis that
aging, and possibly hypertension and its consequences, including cardiac hypertro-
phy, results from actions of AII.
16.1 Angiotensin Receptors
The AT1 receptor belongs to the G-protein-coupled receptor superfamily and has
been cloned and characterized [3, 4]. AT1 receptor is considered to be the mediator
for the cardiovascular and renal effects of AII in normal and, possibly, aging sub-
jects. In contrast, the role, if any, of the AT2 receptor, which is highly expressed
in growing tissues, in the cardiovascular changes in normal and aging subjects,
remains unclear [57]. AII receptors show a great variability and can be detected
in a wide variety of tissues; therefore, it is difficult to relate them to primary hyper-
tension and their role has not been extensively explored in the aging process. Two
AT1 receptor subtypes appear to exist in rodents, the AT1A and AT1B , which exhibit
highly homologous sequences and similar binding and functional characteristics [8].
AT1A expression is by far the dominant form in liver, kidney, vasculature, and heart,
whereas the AT1B is expressed predominantly in the adrenal gland, uterus, and ante-
rior pituitary gland [9]. Both are downregulated by AII.
Complementary mechanisms exist for AII downregulation of the cardiac AT1
gene in vitro via calcium- and cAMP-dependent mechanisms. Such a downregu-
lation of the receptor gene by its agonist is consistent with other members of the
G-protein-coupled family of receptors. The AT2 receptor, however, does not appear
to be G-protein-coupled and possibly signals through phosphotyrosine phosphatase
[10]. AII receptor subtypes have been characterized in cardiac and renal tissue. The
AT1A and AT1B receptors present highly homologous sequences, and similar bind-
ing and functional characteristics [3, 8, 11]. In the rat heart, levels of both AT1 and
AT2 receptor expression are increased during the neonatal period and decrease with
maturation [6, 12].
This family of receptors typically responds to long-term agonist binding with a
decrease in receptor number and mRNA levels [1316]. AII treated mesangial cells
also demonstrate a dose-dependent decrease in AT1 mRNA levels similar to that
seen in cardiocytes and fibroblasts, whereas in some studies AII infused in vivo
had no effect on the AT1 mRNA levels in the rat kidney or aorta, although mRNA
levels were increased in the adrenal gland [17, 18]. By contrast, in models of exper-
imental ureteral obstruction [19, 20], which leads to high intrarenal levels of AII,
AT1 mRNA levels are downregulated, and this downregulation is partially reversed
by angiotensin I converting inhibitors (ACEI) and angiotensin II receptor antago-
nists (AIIRA), suggesting that AII participates in gene regulation and the underlying
pathophysiological conditions may determine the nature of the relationship between
the peptide and the expression of the gene for its receptor. Moreover, in ureteral
obstruction, both glomerular and tubular cells exhibit the changes in AT1 mRNA
levels. This tissue- and cell-specific regulation of the AT1 gene likely speaks to the
potential differences in the role of AII in different segments of various tissues [21].
16 Renin Angiotensin System and Aging 233
The high susceptibility of cardiomyocytes to the effects of AII and aging may
be the result of the density of receptors at this site. Quantitative PCR studies in the
rat have found that both AT1 receptor subtypes are present in the heart with AT1A
mRNA levels threefold higher than AT1B [22].
Analysis of both the number and binding affinity of specific receptors appears
to be important in the understanding of the pathophysiological role of the renin
angiotensin system and in the potential role of AII in the heart and kidney in hyper-
tension and aging. Nevertheless, as already mentioned, variation in AII receptor
expression complicates the design of studies relating it to primary hypertension,
while studies of its potential role in aging are just beginning to emerge [23, 24].
16.2 Angiotensin II and Reactive Oxygen Species in Aging
Experimental findings indicate that hypertension and aging have similar effects on
the structure and function of blood vessels, the heart, and, presumably, the kid-
ney. Both conditions also lead to endothelial dysfunction, decreased vascular com-
pliance, left ventricular hypertrophy, and stiffness. In the normal kidney, despite
no change in blood pressure, AII leads to the expression of genes of sclerosis-
generating cytokines such as transforming growth factor beta (TGF1), platelet-
derived growth factors (PDGF), and osteopontin (OPT), all which enhance tissue
fibrosis [25].
Despite the fact that aging is associated with decreased production and secretion
of renin, kidneys are more sensitive to the renal vasoconstrictive effects of exoge-
nous AII [26], suggesting an increase in receptor number, greater binding affinity
of the peptide, or diminished metabolism of the peptidereceptor complex. Similar
changes might be expected in the aging kidney, which could explain why ACEI or
AIIRA mitigate some of the functional and architectural renal changes observed as
age advances.
AII has been proposed to be a mediator of oxidative stress through the induction
of reactive oxygen species (ROS) in aging and in normal animals [27]. Oxidative
stress is known to induce apoptosis and activation of the pro-inflammatory tran-
scription factor nuclear factor kappa B (NFB), whose biologic effects may play a
role in the renal damage associated with arterial hypertension [28]. Oxidative stress,
fueled in part by AII, upregulates the expression of adhesion molecules, chemoat-
tractant compounds, and cytokines [29].
Mitochondria are a major source of ROS in aging because their mechanisms
of defense from normally formed ROS and superoxide become less effective with
the aging process. Moreover, excessive respiratory chain superoxide can combine
with nitric oxide (NO) produced from mitochondrial nitric oxide synthase (NOS)
to induce apoptosis through the formation of peroxynitrate. This potent oxidant
can promote cytochrome C release by increasing the permeability of mitochondrial
membranes by opening up permeability transition pores [30]. AII contributes to
ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; how-
ever, the mechanism underlying the latter phenomenon is poorly understood. One
234 L.F. Ferder
possibility that has been advanced is that AII activates NADPH oxidase, generating
free radicals that trigger DNA damage and apoptosis [31]. AII blockade can protect
against both age-related mitochondrial dysfunction and ultrastructural alterations,
underscoring the role of RAS in the aging process [27].
It has been suggested that the adaptor protein p66Shc may also be a target for ROS
[32]. When this protein is phosphorylated on Ser36 by oxidative stress, it markedly
sensitizes cells to apoptosis, because it participates in the phosphorylation-induced
repression of Forkhead transcription factors that regulate the expression of various
antioxidant enzymes [32]. Furthermore, activation of a mitochondrial pool of p66Shc
leads to enhanced generation of ROS [33]. Although the study did not examine the
effect of AII on this potential pathway of oxidative damage in aging, it is conceiv-
able that the same mechanism may apply to aging tissues, including the kidney. It
is of great interest that p66Shc knock-out mice were resistant to the deleterious car-
diac effects (remodeling) of sub-pressor doses of AII [34]. It remains to be studied
whether a similar effect of p66Shc occurs in renal tissue.
16.3 Aging Response to AII Blockade
It has long been recognized that plasma renin and aldosterone level fall with advanc-
ing age. Studies in aging animals indicate that both renal renin formation and release
are reduced, and contribute to the fall in plasma renin concentration. The signifi-
cance of this finding has remained obscure and its complexity is enhanced by the fact
that elderly patients develop hypertension, which could be a factor in the reduced
activity of the RAS. Even more intriguing, in attempting to explain the importance
of falls in circulating levels of renin and angiotensin, are the findings reported by
experiments in one of our laboratories.
It was found that CF1 mice treated with the angiotensin I-converting enzyme
blocker Enalapril and Wistar rats treated with an AIIRA exhibited a reduction of
age-associated cardiovascular and renal changes, and had increases in the mitochon-
drial number within cells. This was associated with an increase in survival and lifes-
pan of the animals. The beneficial effect of RAS blockade occurs in spite of the fact
that, as reported, plasma renin concentration and intrarenal renin mRNA is reduced
in older animals [35]. It is, however, consistent with an increase in angiotensin II for-
mation or action within the kidney [36], and supports the concept that the intrarenal
RAS is regulated independently of the circulating system in normal and also in aging
animals.
Our studies revealed that plasma levels of angiotensin I and angiotensin II were
low in control (untreated) aged animals and that, as expected in non-aged normal
animals, aged rats receiving AIIRA exhibited elevated levels of plasma renin, AI and
AII, an indication that the angiotensinrenin feedback mechanism is intact in aged
rats. Aging was accompanied by high glomerular angiotensin II receptor density
as demonstrated by autoradiography. By contrast, receptor density was much lower
in the interstitium of untreated aged animals, and was lower in both glomeruli and
interstitium in the losartan-treated group [37].
Ang II
Receptors AT1 and AT2
OXIDATIVE
Redox Imbalance STRESS Oxidative Damage to MtDNA
Permeability
Transition Pore (PTP) NF-
Activation
Defective Electron
Transport Chain
Profibrotic Cytokines
Release of Cytochrome C
Fibrosis
Apoptosis Energy Deficit
AGING, KIDNEY AND

CARDIOVASCULAR DISEASES
Fig. 16.1 This figure shows how Angiotensin II (All), through oxidative stress, regulates apotosis,
inflammation, and energy balance
These results strongly suggest that the renin angiotensin system plays an impor-
tant role in the mechanisms by which aging affects renal function and that an abnor-
mal regulation of angiotensin II receptors may be responsible for the functional
alterations of the kidney. Moreover, AII effects through the production of reactive
oxygen species, and by damage of mitochondrial function can be responsible for
these alterations. This hypothesis is supported by our findings that ACE inhibitors
and AIIRA decrease tissue oxidative state, improve mitochondrial number and func-
tion, leading to higher animal survival and prolonged lifespan (Fig. 16.1).
16.4 Vascular and Cardiac AII AT1 and AT2 Receptors
The percentages of AT1 and AT2 receptors vary depending on the cell type or tis-
sues considered: in rat aorta 60% of the receptors are AT1 , whereas in total rat
heart, the percentage of AT1 reaches 90% [38] of the angiotensin receptors. Car-
diomyocytes express exclusively the AT1 subtype [39], whereas fibroblasts express
AT2 receptor subtype as well [40]. Although it has been suggested that AT2 recep-
tor activation is involved in the control of cell differentiation, proliferation, and
236 L.F. Ferder
apoptosis [41, 42], the possible roles of AT2 receptors in vivo are poorly under-
stood. In addition, their contribution to the pathophysiology of aging remains
obscure (see below). Moreover, limited knowledge exists of the state of function
and genetic regulation of AT receptors and their subtypes in renal tissues of aging
animals.
AII-induced hypertension and associated cardiac hypertrophy are mediated
mainly via the AT1 receptor subtype. That AII mediates vascular smooth muscle cell
(VSMC) trophic effect via AT2 receptor subtype and independently of a pressure-
dependent mechanism can be inferred from the finding that AT2 receptor blockade
with PD123319 infusion in AII-treated rats had no significant effect on blood pres-
sure but prevented the development of vascular hypertrophy of both aorta and coro-
nary arteries. In addition, in normotensive rats, treatment with losartan alone had no
effect on blood pressure but induced a medial hypertrophy that was prevented by an
additional treatment with PD123319, suggesting that the increase in systemic AII
concentration, as a result of AT1 receptor blockade [43] activates the AT2 receptors
and, as a consequence, unmasks their trophic effect. The trophic effect of the AT2
receptor subtype appears to be specific to VSMCs and independent of whether it is
found in conductive (aorta) or resistance (coronary artery) vessels. Also, it has been
shown that AT2 receptors play a major role in myointimal formation after arterial
injury [44] and that the expression of AT2 receptors is increased in hypertrophied
left ventricle [45]. The role, if any, of this interactive regulation between AT1 and
AT2 receptors in aging tissues (whether accompanied or not by hypertension), par-
ticularly the kidney, remains to be established.
The relative abundance of AT2 subtype receptors in many fetal tissues supports
a role of AT2 during development [46]. Furthermore, a key role for the AT2 sub-
type receptor is suggested by several studies that correlate enhanced AT2 receptor
expression with cardiovascular system disease states such as diabetes, hypertension,
and senescence [4749]. The ability of a tissue to change the expression of AT1
receptors to AT2 has been described in experimentally induced vascular injury [21],
suggesting that AII may also play a role, through AT2 receptors, in smooth mus-
cle cell differentiation and proliferation. In addition, Brilla et al. [40] reported that
in cultured adult rat cardiac fibroblasts AII stimulates collagen synthesis by both
AT1 and AT2 receptors, and that AII inhibition of collagenase activity is specifically
mediated by the AT2 subtype receptor.
Chronic pharmacological blockade of AT2 subtype AII receptors has no systemic
hemodynamic (arterial pressure, cardiac output, heart rate) either in normotensive
rats or in AII-induced hypertensive rats, and does not affect the hypertension-
induced cardiac hypertrophy [50]. Furthermore, plasma angiotensin II level and aor-
tic reactivity to AII are not affected after chronic AT2 receptor blockade. Chronic
blockade of AT2 receptors antagonizes the vascular growing effects related to long-
term AII injection, whereas blockade of AT1 receptors does not. The vasotrophic
effect of AII is at least partially mediated via AT2 receptor subtype in some experi-
mental model of hypertension.
Considering AIIs role in the induction of cardiac hypertrophy and possibly fibro-
sis of tissues, such a mechanism to decrease receptor number could be essential
for maintaining normal cardiac structure and function. A similar proposal could be
marshaled for the kidney. The protective effects of AII blockade, by either dimin-
ishing its production or its actions, on fibrosis and proliferation is indirect evidence
that, despite low angiotensin II production, increased sensitivity of the kidney to the
effects of the hormone could mediate the development of fibrosis and tissue damage
in aging, hypertension, or aging accompanied by hypertension.
16.5 Aging and Enhanced Cardiac Expression of Angiotensin II

Receptor Subtypes
In the heart and the kidney, the presence of angiotensinogen (ANG), renin, and
angiotensin-converting enzyme (ACE) suggests local synthesis of AII [51, 52]. In
young adult rats, the myocardium is able to synthesize AII from angiotensinogen
and angiotensin-converting enzyme [51], but it has been shown that a several-fold
increase in both ANG and ACE mRNA occurs in the aged LV but not in the RV.
This suggests that during aging, an activation of cardiac AII synthesis may com-
pensate in part for the depression of the circulating RAS. The possibility cannot be
discarded that a similar mechanism exists to maintain normal intrarenal resistances
and glomerular filtration fraction in the aging kidney.
Although less is known relative to the kidney, senescence is associated with
marked changes in cardiac structure and morphology such as cardiomyocyte loss,
hypertrophy of the remaining cells, and the development of fibrosis [53, 54]. These
changes may account for the functional characteristics of the senescent myocardium
such as impaired myocardial perfusion [55], altered diastolic compliance [53], and
arrhythmias [56]. Vascular structures are also modified, and aging is associated with
increased arterial wall stiffness, as shown by a significant decrease in systemic and
local arterial compliance and an increase in aortic impedance [53, 57], which may
induce mild left ventricular hypertrophy. There is now evidence that senescence
is associated with increased cardiac ANG and ACE gene expression, suggesting
increased cardiac AII synthesis, whereas plasma RAS activity, as already men-
tioned, is largely depressed [58].
The presumed mechanistic pathways involved in the increase in cardiac AII
receptor gene expression are multiple. Upregulation could be intrinsic to the devel-
opmental gene reprogramming often associated with senescence. Cardiac senes-
cence is characterized by the re-expression of fetal proteins, such as the contractile
protein isoform b-MHC and the atrial natriuretic peptide gene [59, 60]. On the other
hand, several studies in rat ventricular tissue have demonstrated developmental reg-
ulation of cardiac AII receptor subtype densities and gene expression, which are
abundant during the neonatal period and decrease with maturation [6163]. Sec-
ondly, humoro-hormonal status is modified during senescence and is characterized
by a large decrease in plasma AII synthesis and an increase in plasma cortisol level
[58]. A number of circulating factors, such as vasoactive substances, growth factors,
and steroids, modulate AT1 expression via their effects on the transcriptional activity
of AT1 gene [18, 64, 65]. However, the differential pattern of AT1 gene expression
observed in the LV and RV of aged rats makes a major role of these hormones in the
regulation of AT1 gene expression unlikely.
238 L.F. Ferder
Much less is known about the hormonal regulation of AT2 mRNA level. However,
based on the upregulation of AT2 gene expression in both ventricles of aged rats,
hormonal and humoral factors might be one of the triggers for the increase in car-
diac AT2 gene expression during senescence. Mechanical factors are repeatedly pro-
posed as triggers for the regulation of genetic expression during the development
of cardiac hypertrophy. Activation of AT1 and/or AT2 gene expression has been
demonstrated in ventricular tissue during hemodynamic overload [66, 67]. Mechan-
ical stretch has also been recently shown to upregulate AT1 and AT2 gene expression
in neonatal rat cardiac myocytes, the increase in AT1 gene expression being mainly
due to increased transcription, whereas that of AT2 results from stabilization of AT2
mRNA metabolism [68]. Even though cardiac output and ejection fraction of both
aged human and rat hearts are unaltered, the increase in vascular stiffness and aor-
tic impedance during aging result in a moderate increase in LV afterload [69, 70].
These changes in LV properties might therefore account, at least in part, for the
upregulation of AT1 gene expression in the LV of aged rats.
Heymes and collaborators [49] have demonstrated increased density of both AT1
and AT2 receptors in the LV myocardium of senescent rats and activation of the
intracardiac RAS in aging, associated with suppression of plasma AII synthesis.
Such local and independent regulation of intracardiac AII synthesis and receptor
subtype expression could account for both autocrine and paracrine actions [71] and
support the concept of intracardiac AII production as a regulator of cardiac hypertro-
phy and collagen accumulation, a condition that could also prevail in renal tissues.
A strong correlation between AT2 gene expression and fibrosis in both ventricles,
compared with the correlation of AT1 and ACE mRNA levels with LV fibrosis only,
has suggested [49] that age-associated cardiac fibrosis is more closely related to the
AT2 than the AT1 receptor subtype. This suggestion is given credence by studies
of Lorell et al. [72], who demonstrated that, both AT1 inhibition and decrease in
ACE gene expression affected neither cardiac fibrosis nor hypertrophy in a pressure-
overload rat model.
Upregulation of the cardiac RAS or the intrarenal RAS may, in part, compensate
for the large age-related fall in plasma AII synthesis. However, activation of local
RAS systems and increased expression of AII receptor subtypes might have detri-
mental effects when other pathological manifestations often associated with senes-
cence, such as hypertension, heart failure, tubulointerstitial or glomerular damage,
are superimposed. AII receptor subtype antagonists could therefore be therapeuti-
cally useful in normal elderly people or elderly people with renal disease.
16.6 Kidney AII Receptors as a Function of Age
Studies by Correa and collaborators [73] in immature (1-week-old) and adult (12-
week-old) normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats
(SHR) revealed that AII receptors and ACE binding sites, measured by quantita-
tive autoradiography for quantification of AII receptors in both neonatal and adult
animals of either strain, were of AT1 subtype. In all kidney segments of 1-week-old
rats, AII receptor density was higher in SHR than WKY. Binding density increased
with age in WKY rats; thus, in the glomeruli and the outer stripe of the outer
medulla of 12-week-old WKY, binding was significantly higher than that present
in age-matched SHR. By contrast, [125 I]351A (an iodotyrosyl derivative of the ACE
inhibitor lisinopril that binds specifically to the active site of ACE [74]) was highest
in the outer medulla and not detectable in glomeruli. In 1-week-old rats, binding to
ACE was higher in WKY than in SHR strain. Differences in ACE binding between
adult SHR and WKY rats were inexistent, with the exception of the inner stripe of
the outer medulla, where no binding was detected in SHR. These studies suggest
that the renal RAS is developmentally regulated and is involved in the genesis and
maintenance of genetic hypertension in SHR as aging proceeds. The evolution of
the regulation of ACE and the AT1 and AT2 receptor beyond 12 weeks of aging
and how this evolves in relation to hypertension and renal damage remain to be
studied.
An influence of age on the RAS can also be gleaned from studies in male het-
erozygous transgenic hypertensive rats TGR (mREN2)27 (TGR) [75]. As compared
to Sprague-Dawley control rats, receptor density was significantly lower in TGR.
Measurement of density and affinity of AII receptors in glomeruli of animals 11
weeks old as compared to 1820 weeks old rats revealed that receptor number
increased with aging. In renal arteries, the AII receptor mRNA of the main recep-
tor subtype AT1A was neither strain- nor age-dependent, AT1B - and AT2 -receptor
mRNAs were significantly lower in TGR than SPRD rats. This study provide
evidence that an overactive renin angiotensin system in TGR rats led to a down-
regulation of glomerular angiotensin II receptors that was not accompanied by a
downregulation of the mRNA of the dominant AT1A -receptor subtype, particularly
as age advanced. Diminished density of AII receptor leading to enhanced sensitivity
to AII could explain in part the development of nephrosclerosis and the tubulointer-
stitial damage seen in these rats, particularly as they age.
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Subject Index
A Ang II receptor blockade

ACE2 clinical efficacy in hypertension, 62,
and cardiac arrhythmias, 3, 85 6970
and enzyme regulation in heart, 123125 pleiotropic actions, 7172
ACE inhibitors AT1 receptor blockade
and B2 kinin receptors, 161, 163, and activation of autoantibodies,
164, 166 6365, 66
and kinin B1 receptors, 133135 ang II-independent activation of AT1
Adipose tissue, 16, 193 receptor, 6263
and adipokines, 186187, 189 and cross-talk, 6768
Adrenal RAS, 29 and dimerization, 6768
Aging and G protein interacting proteins,
ang Ang II blockade, 227, 230231, 233 6567
and cardiac expression of Ang II receptors, and hypertensive disease, 5974
233234 renal AT1 receptors and blood flow, 68
and kidney Ang II receptors, 234235 AT1 receptors, 3, 35, 36, 37, 38, 39, 40, 45,
and RAS, 227235 46, 47, 5974, 8384, 88, 106, 107,
and reactive oxygen species, 227, 108, 109, 110, 111, 112, 113, 114,
229230, 231 122, 124, 167, 168, 206, 208, 212,
Aldosterone 213, 214, 215, 216, 217, 218, 219,
and Ang II, 205 220, 222, 228, 229, 232, 234
and arrhythmogenic effects, 206 chronic blockade, 8384, 232
biosynthesis, 60, 204205 AT2 receptor activation, 212, 231
and fibrogenic effects, 206 and AT1 receptor blockade, 232
receptor, 87, 112, 204 Atherosclerosis
signaling pathways, 37, 38, and ACE, 40, 46, 153, 216
206207, 208 and AT1 receptor blockers, 213, 218
and vascular effects, 41, 205 and AT2 receptor upregulation,
Ang (1-12), 125 220, 221
Ang (1-7) and collagen deposition, 221, 222
and cardiac arrhythmias, 85, 121 and gene therapy/AT2 overexpression,
and cardiac dynamics, 121 219220
and cell volume, 8587 and renin inhibitors, 217218
and ischemia/reperfusion, 85, 121
Ang II C
and aldosterone, 36, 42, 47, 190, 191 Cell volume, 3, 8587
and oxidative stress, 38, 39, 41, 47, 71, 83, and RAS, 8587
8485, 188, 189, 190, 191, 192, Chymase, 2, 26, 27, 29, 72, 95, 111,
205, 212218, 221, 231 215, 216
Disease, Contemporary Cardiology, DOI 10.1007/978-1-60761-186-8,

246 Subject Index
CMS Kinins
and HTN, 184, 186, 187, 188, 191, 193, and ACE inhibitors, 131141, 148, 149,
195, 196 153, 154, 155, 158, 160167,
and hyperinsulinemia, 186, 187188, 196 168, 169
and therapeutic approach, 193196 and cardiovascular diseases, 147169
Components of RAS, 1, 2, 3, 28, 31 and NO, 133135, 140, 148, 149, 151,
152, 153, 161, 163, 165, 166, 167,
D 168, 169
Diabetes and water and electrolytes, 155157
and RAAS
and endothelium, 190191 L
and heart, 190, 194, 195 Local renin angiotensin system
and kidney, 191192 and clinical inclications, 712
and pancreas, 192193, 195 and ventricular hypertrophy, 103114
Dyslipidemia, 184, 186, 187, 188, 193, 195,
213, 214, 218, 222 O
Oxidative stress
E and insulin resistance, 186, 188, 189,
Eplerenone 195, 196
and AT1 receptors, 3, 46, and local RAAS, 186, 188, 189, 196
88, 208
and intracrine RAS, 88 P
Prorenin
F and diabetes, 3, 16, 17, 19, 20, 22, 26, 27,
Failing heart 28, 30
connexins, 82 and retinopathy, 3, 16, 17
remodeling, 8188 (Pro) Renin receptor
reprogramming, 82, 85 biochemistry, 1820
and cardiovascular disease, 20, 22
I conformation change, 18
Intracellular Ang II intracellular renin binding
on calcium current, 2, 3 protein, 18
on cell communication, 86 M6P/IGF2 receptor, 18
Intracrine renin angiotensin aldosterone system and MAP kinases, 1722
(RAAS) ontogeny, 2122
and cardiovascular diseases, 208 and renal disease, 20
and diabetes, 99, 183196
and uterus and pregnancy, 11 R
RAAS inhibition
K and heart failure, 45, 46, 99
Kallikrein-kinin system and myocardial infarction, 46, 99
and angiotensin II receptor blockers, RAAS, see Intracrine renin angiotensin
167169 aldosterone system
and blood pressure, 140, 154, 157, 158, RAS, 1, 2, 3, 20, 21, 25, 26, 27, 2831, 41, 42,
160, 163, 167 46, 47, 59, 61, 62, 63, 64, 65, 68,
and heart failure, 165167, 169 69, 71, 72, 73, 74, 82, 83, 84, 86,
and local blood flow, 153154 88, 104108, 109114, 119, 120,
and LV hypertrophy, 163 122, 123, 125, 126, 155, 186, 192,
and myocardial ischemia, 164165 227, 230, 233, 234, 235
and renal blood flow, 154155, 157 and atherosclerosis, 211223
Kininases, 132, 135, 136, 140, 149, 151, 153, Renal RAS, 2930
156, 157 Renin, 13, 712, 1522, 2531, 3548, 59,
Kininogens, 140, 149, 150, 151, 152, 153, 155, 60, 64, 68, 69, 73, 74, 82, 83, 84,
156, 157, 158, 160, 166, 167, 168 8587, 93100, 104, 112, 113,
Subject Index 247
114, 119126, 131, 133, 140, 148, S

153, 154, 155, 156, 159, 160, 161, Salt, 9, 1011, 27, 35, 36, 39, 42, 43, 71, 106,
163, 167, 168, 183196, 203209, 121123, 148, 157, 158, 159, 160,
211223, 227235 169, 185, 186, 190, 191, 204, 205
gene expression, 26, 28
and ACE2/Ang (17)/mas Axis, 121123
Renin transcript, 2, 29, 31
overexpression, 2 Salt-loading, 1011
Renin uptake, 29 and cardiovascular mortality, 10, 11

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Contemporary Cardiology

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Renin Angiotensin System

ISBN 978-1-60761-185-1 e-ISBN 978-1-60761-186-8

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1 Systemic Versus Local Renin Angiotensin Systems.

10 Angiotensin-(1-7), Angiotensin-Converting Enzyme 2,

Sarfaraz Ahmad, MD, PhD Hypertension and Vascular Research Center,

Changping Hu, MD, PhD Division of Cardiovascular Medicine, University of

Bernard Swyngedauw, MD, PhD Centre de Recherches Cardiovasculaires

Walmor C. DeMello and Richard N. Re

Abstract The concept of local renin angiotensin systems in the cardiovascular

W.C. DeMello (B)

Abstract The concept of local renin angiotensin (and possibly aldosterone)

2.1 The Classical System

E.D. Frohlich (B)

protein angiotensinogen which is synthesized in the liver. The consequence of this

2.2 The Local Systems

as hemodynamic factors [24, 25]. These provocative findings permitted a further

2.3 Structural and Function Response of Organs to Salt-Loading

Abundant clinical and experimental evidence has accumulated in recent years to

2.4 Secondary Organ Responses of Therapy

2.5 Toxemias of Pregnancy

may be related to inadequate perfusion of the utero-placental unit and/or a conse-

Genevieve Nguyen and Aurelie Contrepas

(pro)renin: designate renin and prorenin

3.2 Renin and Prorenin

The term renin is used to cover two entities:

renin, the mature enzyme which is catalytically active in solution and

3.3 The (Pro)Renin Receptor

3.3.1 Biochemistry of the (P)RR

The (pro)renin receptor is a 350-amino acid receptor with a single transmembrane

3.3.2 (P)RR in Experimental Models of Cardiovascular

3.3.3 Unexpected Properties and Ontogeny of the (P)RR

Analysis of the sequence of (P)RR-coding cDNA shows that sequence coding

Abstract The renin angiotensin system (RAS) is an established regulator of

angiotensin signalling in the environment of high-circulating oxidized LDL [11]. In

4.2 Studies in Man

production of angiotensin II results in increased renovascular resistance and renin

4.3 Studies in Animal Models

4.3.1 Cardiac RAS

4.3.2 Adrenal RAS

4.3.3 Renal RAS

4.3.4 Intracellular RASs

renin antisense, again indicating the existence of an intracellular RAS [5759].

Pierre Paradis and Ernesto L. Schiffrin

Abstract The renin-angiotensin-aldosterone system (RAAS) plays a critical role

Classically, the renin-angiotensin-aldosterone system (RAAS) was described as

E.L. Schiffrin (B)

5.1 Blood Vessels

Essential hypertension is characterized by increased peripheral vascular resistance

tance by augmenting vasculature stiffness [47]. Vascular fibrosis is characterized by

contribute to different degrees. Increase in ROS was documented in humans with

which leads to platelet activation and thrombus formation. Plasminogen activator

5.2 The Kidney

5.3 The Heart

specific cardiomyocyte AT1 receptor overexpression revealed an increased

5.4 The Brain and the Sympathetic Nervous System

In addition to the brain, Ang II induces increased release of norepinephrine (NE)

cells by a transforming growth factor-beta-independent mechanism. Circulation 111(19),

Abstract The renin angiotensin system (RAS) is a coordinated hormonal cascade,

R.M. Carey (B)