Download Angiotensin-converting enzyme in cardiovascular function and dysfunction Liza Ljungberg

Linköping University Medical Dissertations No 1224
Angiotensin-converting enzyme in
cardiovascular function and dysfunction
Liza Ljungberg
Division of Cardiovascular Medicine
Department of Medical and Health Sciences
Faculty of Health Sciences
Linköping University, Sweden
Linköping 2011
 Liza Ljungberg, 2011
Cover: 3D structure of the extracellular/soluble part of angiotensin-converting
enzyme obtained from Entrez´s 3D structure database MMDB (ID84704).
Published articles have been reprinted with permission from the copyright
holder.
During the course of the research underlying this thesis, Liza Ljungberg was
enrolled in Forum Scientium, a multidisciplinary doctoral program at Linköping
University, Sweden.
Printed in Sweden by LiU-tryck, Linköping, Sweden, 2011
ISBN 978-91-7393-243-1
ISSN 0345-0082
The scientist is not a person
who gives the right answers,
he is one who asks the right questions
Claude Lévi-Strauss
ABSTRACT
Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin
system, converting angiotensin I to the vasoactive peptide angiotensin II, and
degrading bradykinin. Angiotensin II is a multifunctional peptide, acting on a
number of different tissues. A common genetic variation in the gene encoding
ACE; ACE I/D polymorphism influences the level of ACE in the circulation, and has
been linked to increased risk for cardiovascular disease. This thesis aimed to
explore the connection between ACE and cardiovascular function and
dysfunction.
The impact of nicotine and nicotine metabolites on ACE in cultured human
endothelial cells was studied. Nicotine as well as nicotine metabolites induced
increased ACE activity in cultured human endothelial cells. In elderly men higher
ACE levels were seen in smokers compared to non-smokers. Furthermore,
diabetes was associated with higher circulating ACE. Increased ACE level may
represent a cellular mechanism which contributes to vascular damage.
Elderly men carrying the ACE D allele had higher abdominal aortic stiffness
compared to men carrying the I/I genotype. Our data suggest that the mechanism
by which the ACE D allele modulates aortic wall mechanics is independent of
circulating ACE levels. Previous studies have indicated a link between the D allele
and abdominal aortic aneurysm. Increased aortic stiffness suggests impaired
vessel wall integrity, which combined with local hemodynamic and/or
inflammatory factors may have a role in aneurysm formation.
Subjects with left ventricular dysfunction had higher levels of circulating ACE
compared to those with normal left ventricular function, while there was no
association between ACE and central hemodynamics. ACE might play a role in the
pathogenesis of left ventricular dysfunction and our findings suggest a direct
effect on the heart rather than affecting central blood pressure.
POPULÄRVETENSKAPLIG SAMMANFATTNING
Renin-angiotensin-aldosteron systemet är ett av kroppens viktigaste system för
reglering av blodtryck. Angiotensin-converting enzyme (ACE) utgör en central del
i renin-angiotensin-aldosteron systemet genom att omvandla den inaktiva
peptiden angiotensin I till den betydligt mer aktiva peptiden angiotensin II.
Angiotensin II har en rad olika effekter i olika vävnader och är involverad i
regleringen av kardiovaskulära systemet, och sannolikt även i utvecklingen av
hjärt-kärlsjukdom. Hos friska individer finns ACE lokaliserat i det innersta
cellagret (endotelet) i blodkärlens väggar, samt även fritt cirkulerande i blodet.
Det finns en vanlig genetisk variation (polymorfi) i genen för ACE som brukar
benämnas ACE I/D. Den styr till viss del hur mycket ACE som finns cirkulerande i
blodet, även om det finns en stor variation även hos individer med samma genvariant. Tidigare studier har indikerat att ACE I/D polymorfin kan öka risken för
hjärt-kärlsjukdom. I de fyra delarbetena som ligger till grund för denna
avhandling studerades relationen mellan ACE och kardiovaskulär funktion samt
sjukdom i kardiovaskulära systemet.
Det är välkänt att rökning har negativa effekter på hjärta och kärl, och nikotin
verkar kunna påverka flera processer som är involverade i uppkomst av hjärtkärlsjukdom. När nikotin tas upp i blodet omvandlas (metaboliseras) det
framförallt i levern och ett antal nya metaboliter bildas. Trots att metaboliterna
förkommer i hög koncentration i blodet hos tobaksnyttjande individer, är deras
påverkan på hjärta och kärl relativt okänd. I delarbete I användes mänskliga
endotelceller isolerade från blodkärl i navelsträngar, för att studera hur nikotin
och levermetaboliter av nikotin påverkar ACE. En ökad aktivitet av ACE, och
därmed ökad bildning av angiotensin II skulle kunna vara en cellulär mekanism
som leder till ökad risk för hjärt-kärlkomplikationer. Både nikotin och flera av
nikotinmetaboliterna visade sig ökar ACE aktiviteten. Nikotin och dess
metaboliter hade olika stor effekt på ACE i celler från olika navelsträngar. Det är i
dagsläget oklart vad denna individuella skillnad beror på, men våra resultat tyder
på att det verkar vara oberoende av ACE I/D polymorfin.
Ett flertal riskfaktorer för hjärt-kärlsjukdom har identifierats (t.ex. rökning och
diabetes), men man har inte lyckats kartlägga de bakomliggande mekanismerna
för uppkomsten av hjärt-kärlsjukdom. I delarbete II studerades sambandet
mellan ACE I/D polymorfin, mängd ACE i blodet, kardiovaskulära riskfaktorer
samt förekomst av hjärt-kärlsjukdom i en population av 672 äldre män och
kvinnor. Hos män fanns ett samband mellan antalet riskfaktorer och mängd ACE i
blodet. Rökning och diabetes var de riskfaktorer som visade sig ha störst
betydelse för mängden ACE i blodet. Det är tänkbart att en ökad mängd ACE är en
av de cellulära mekanismer som leder till ökad risk för kardiovaskulära
komplikationer hos rökare och diabetiker.
I 406 individer av populationen som studerades i delarbete 2 undersöktes även
mekaniska egenskaper (styvhet/elasticitet) i den stora kroppspulsådern i buken
(bukaorta). Detta område är speciellt ofta drabbat av sjuklig vidgning med ett
pulsåderbråck som resultat. De bakomliggande mekanismerna för utveckling av
pulsåderbråck är okända, men man tror att det finns genetiska variationer som
ger en ökad benägenhet att utveckla pulsåderbråck. ACE I/D polymorfin är en av
de gen-variationer som pekats ut som en möjlig kandidat. I det tredje delarbetet
studerades kopplingen mellan ACE och mekaniska egenskaper i bukaorta. Män
som var genetiska bärare av ACE D varianten visades ha en högre styvhet i
bukaorta, medan inget sådant samband kunde återfinnas hos kvinnor. Inget
samband mellan mängd ACE i blodet och styvhet i bukaorta kunde identifieras,
varken hos kvinnor eller hos män. En förändrad väggmekanik indikerar en störd
funktion i aortaväggen som tillsammans med andra faktorer skulle kunna ge en
ökad risk för utveckling av pulsåderbråck hos män som är bärare av D varianten
av ACE genen.
Ett högt blodtryck är en välkänd riskfaktor för att utveckla hjärt-kärlsjukdom.
Vanligen mäts blodtycket i överarmen, men på senare år har det centrala
hjärtnära blodtrycket i aorta kommit mer i fokus, eftersom detta tryck visat vara
mer kraftfullt relaterat till utveckling av hjärt-kärlsjukdom. I delarbete IV
studerades sambandet mellan ACE, centralt blodtryck och hjärtfunktion på äldre
män och kvinnor i en delpopulation av individerna i delarbete II. De med nedsatt
vänsterkammarfunktion i hjärtat hade högre mängd ACE i blodet jämfört med
individer med en normal hjärtfunktion. Däremot fanns inget samband mellan
mängd ACE och blodtryck. ACE I/D polymorfin hade ingen effekt på varken
hjärtfunktion eller blodtryck. ACE skulle kunna ha en direkt påverkan på hjärtat
snarare än indirekt via påverkan på blodtrycket vid nedsatt
vänsterkammarfunktion.
TABLE OF CONTENTS
ABBREVIATIONS
1
BACKGROUND
5
LIST OF PAPERS
The renin-angiotensin-aldosterone system
Angiotensin-converting enzyme
Structure, location and substrates
The ACE gene
Regulation of ACE
Nicotine and nicotine metabolites
Cardiovascular effects of nicotine
3
5
7
7
8
9
11
12
The abdominal aorta
13
Heart failure and RAS
16
Arterial stiffness
Arterial pulse wave and wave reflection
AIMS
MATERIALS AND METHODS
14
15
17
19
Cell culturing, HUVECs (paper I)
19
Determination of ACE level
21
Study populations (paper II-IV)
Determination of ACE activity
20
21
Validation of ACE assays
22
Determination of abdominal aortic wall mechanics
25
Stability of ACE during storage at -70°C
ACE genotyping
Central aortic hemodynamics
Echocardiographic evaluation of left ventricular function
RESULTS AND DISCUSSION
Effect of nicotine and nicotine metabolites on ACE in vitro
23
24
27
28
29
29
Individual differences in the response to nicotine and nicotine
metabolites
Association between ACE and cardiovascular risk
ACE level – influence of ACE genotype, age and gender
ACE level and cardiovascular risk factors
ACE inhibitor treatment
Association between ACE and abdominal aortic wall mechanics
ACE I/D polymorphism and abdominal aortic wall mechanics
Circulating ACE and arterial stiffness
ACE, abdominal aortic stiffness and aneurysm
Association between ACE, left ventricular function and central
hemodynamics
32
34
34
35
37
38
38
39
40
42
LIMITATIONS
46
ACKNOWLEDGMENTS
49
CONCLUSIONS
REFERENCES
47
53
ABBREVIATIONS
AAA
ACE
ACEi
AIx
AIx HR75
Ang I
Ang II
ARBs
AT1
AT2
CC
CVD
D
DC
D/D
HUVECs
I
I/D
I/I
IMT
LV
LVH
PCR
PWV
RAAS
RAS
VEGF
Abdominal aortic aneurysm
Angiotensin-converting enzyme
Angiotensin-converting enzyme inhibitors
Augmentation index
AIx adjusted to a heart rate of 75 beats/minute
Angiotensin I
Angiotensin II
Angiotensin II receptor blockers
Angiotensin II receptor 1
Angiotensin II receptor 2
Compliance coefficient
Cardiovascular disease
Deletion
Distensibility coefficient
Deletion/deletion
Human umbilical vein endothelial cells
Insertion
Insertion/deletion
Insertion/insertion
Intima-media thickness
Left ventricular
Left ventricular hypertrophy
Polymerase chain reaction
Pulse wave velocity
Renin-angiotensin-aldosterone system
Renin-angiotensin system
Vascular endothelial growth factor
ABBREVIATIONS I 1
LIST OF PAPERS
This thesis is based on the following papers, which will be referred to by their
roman numbers.
I.
Effect of Nicotine and Nicotine Metabolites on Angiotensin-Converting
Enzyme in Human Endothelial Cells
Liza U Ljungberg, Karin Persson, Endothelium 2008:15(5):239-245
II.
The association between circulating angiotensin-converting enzyme
and cardiovascular risk in the elderly – a cross sectional study
Liza U Ljungberg, Urban Alehagen, Hanna M Björck, Toste Länne, Rachel
De Basso, Ulf Dahlström, Karin Persson, JRAAS, published online 27
January 2011 (DOI:10.1177/1470320310391326)
III.
Impaired abdominal aortic wall integrity in elderly men carrying the
angiotensin-converting enzyme D allele
Liza U Ljungberg, Rachel De Basso, Urban Alehagen, Hanna M Björck, Karin
Persson, Ulf Dahlström, Toste Länne (submitted manuscript)
IV.
Circulating angiotensin-converting enzyme levels are associated with
left ventricular dysfunction, but not with central aortic blood
pressure, aortic augmentation or pulse pressure amplification
Liza U Ljungberg, Urban Alehagen, Rachel De Basso, Karin Persson, Ulf
Dahlström, Toste Länne (submitted manuscript)
LIST OF PAPERS I 3
BACKGROUND
The renin-angiotensin-aldosterone system
The renin-angiotensin-aldosterone system (RAAS) is a powerful system
regulating fluid-electrolyte balance and systemic blood pressure. A schematic
overview of RAAS is shown in Figure 1.
Endothelium
Liver
ACE
Brain: Sympathetic activity
Vasopressin secretion
Thirst and salt appetite
Vessels: Vasocontriction
Remodeling
Angiotensinogen
Angiotensin I
Renin
Kidney
Angiotensin II
Adrenal gland: Aldosterone secretion
Kidney: Na+ Ca2+ reabsorption
K+ excretion
H2O retention
Heart: Inotropy
Hypertrophy
Fibrosis
Figure 1. Schematic overview of the renin-angiotensin-aldosterone system [1].
ACE: Angiotensin-converting enzyme
Angiotensinogen is constitutively produced and released from the liver into the
circulation where it is converted to angiotensin I (ang I) by renin. Renin is a
proteolytic enzyme synthesized, stored and released primarily from the
juxtaglomerular apparatus in the kidneys in response to decreased blood
pressure, low sodium concentration and increased sympathetic activity. Ang I has
mild vasoconstrictor properties but not enough to cause significant physiological
effects. Ang I is further converted to angiotensin II (ang II) by angiotensinconverting enzyme (ACE). Although ACE is the major catalyst for this conversion,
other enzymes are also able to generate ang II e.g. chymase [2], cathepsin [3] and
tonin [4]. Ang II induces negative feedback inhibiting the release of renin, thereby
acting in a self-regulatory manner. The renin-angiotensin system (RAS) was
originally considered as an endocrine system. However, during the last decades it
BACKGROUND I 5
has become evident that all components of RAS (i.e. renin, angiotensinogen, ang I,
ang II, ACE and angiotensin receptors), are present in a number of different
tissues, including the vessel wall, the heart, and the kidneys [5].
Ang II is considered the main effector peptide of RAS. Ang II induces its effects
through binding to specific receptors [6]. Most of the physiological effects are
generated through the AT1 receptor [6], while the AT2 receptor is not as well
characterized, but may counteract some of the processes mediated by the AT1
receptor [7, 8].
Ang II is a multifunctional peptide, acting on a number of different tissues (Figure
1). Ang II stimulates release of aldosterone from the adrenal gland and generates
constriction of renal arterioles thereby increasing salt and water retention in the
kidneys. In the brain, ang II is involved in regulation of salt-and fluid homeostasis
by influencing the autonomic nervous system [9], vasopressin release [10] as well
as thirst and salt appetite [11]. In the blood vessels, ang II act as a powerful
vasoconstrictor, and is involved in vascular wall remodeling through induction of
smooth muscle cell growth [12, 13], up-regulation of growth factors [14] and by
affecting the synthesis of extracellular matrix proteins [15, 16]. In the heart, ang II
has inotropic [17, 18] and hypertrophic effects [19] and promotes cardiac fibrosis
[20]. In addition, ang II seems to be involved in inflammation by inducing upregulation of endothelial adhesion molecules [21-23], proinflammatory cytokines
[24, 25], and production of reactive oxygen species [25-27].
Degradation of ang II into other peptides occurs rapidly after its formation [28]. A
number of angiotensin peptides have been identified during the last decades, and
named according to number of amino acids [29]. Angiotensin 1-7 has attracted
the most attention as it has been shown to be pharmacologically active [30-32],
and as it counteract some of the effects caused by ang II [33]. The conversion of
angiotensin 1-9 into angiotensin 1-7 [34], as well as degradation of angiotensin
1-7 to angiotensin 1-5 is catalysed by ACE [35].
6 I BACKGROUND
Angiotensin-converting enzyme
Structure, location and substrates
ACE is a 170 kDa Zn2+ metallopeptidase existing in two isoforms. Somatic ACE is
expressed in various tissues and cell types including the cardiovascular system,
kidneys, intestine, adrenal glands, liver, uterus etc [36]. Testicular ACE, on the
other hand, can only be found in germinal cells in the testes [37]. In the
cardiovascular system, ACE exists both in a soluble form in the blood and bound
to the cell membrane of different cell types [38-40]. High levels of ACE can be
found in vascular endothelial cells [38], but has also been identified in e.g. Tlymphocytes [39], macrophages [40], in cardiac tissue [41], and in the vessel wall
[42]. In endothelial cells, ACE is located on the luminal side. The C-terminal part is
anchored to the plasma membrane [43], with a hydrophobic trans-membrane
domain and a short cytoplasmic fragment (Figure 2). The extracellular part
consists of two homologous domains, with two catalytic sites and Zn2+ binding
regions [43, 44].
Active site
Active site
NH2
Secretase
Plasma membrane
COOH
Figure 2. ACE located on the luminal side of vascular endothelium. ACE has two
homologous domains with two active sites. The N-terminal part is released into the
circulation following cleavage of the C-terminal domain by secretase.
Circulating ACE originates mainly from endothelial cells and is released into the
blood after proteolytic cleavage of the anchor [45, 46]. One of the major sources
for production of ACE is the lungs, due to its high vascularisation. If the
circulating level of ACE reflects the level of ACE in tissues is still unknown.
BACKGROUND I 7
Besides catalyzing the conversion of ang I to ang II, ACE also acts on a number of
other natural substrates e.g. bradykinin [47], enkephalin [48], neurotensin [49]
and substance P [49].
The ACE gene
The gene encoding ACE is located on chromosome 17q23. This gene encodes both
ACE isoforms, but has two different promoters resulting in different mRNAs [50].
There is a common genetic variation within the gene, consisting of a 287 bp
insertion/deletion (I/D) polymorphism located in intron 16 [51] (Figure 3). This
polymorphism generates three different ACE genotypes: I/I, I/D and D/D.
Deletion allele, D
exon
exon
exon
Insertion allele, I
exon
287 bp
exon
exon
Figure 3. Schematic illustration of the 287 bp insertion/deletion polymorphism
located in intron 16 within the ACE gene.
As the ACE I/D polymorphism is located within an intron it will not affect the
structure of the enzyme. However, there is an association between the ACE I/D
polymorphism and circulating levels of ACE, where I/I, I/D and D/D genotypes
have low, medium and high levels, respectively [51]. The ACE I/D polymorphism
has been shown to account for approximately 20-50% of the variation in plasma
ACE [51, 52]. The influence of the ACE I/D polymorphism on ACE level does not
seem to be restricted to the circulation as similar patterns have been detected in
e.g. T-lymphocytes [39] and cardiac tissue [41], suggesting that tissue ACE and
circulating ACE are under similar genetic control.
Given the fact that the ACE I/D polymorphism is located in a non-coding region, it
is unlikely that this is the functional variant responsible for the differences in
8 I BACKGROUND
circulating ACE. A strong genetic linkage has been shown in the chromosomal
region in which the ACE gene is located [53, 54], suggesting that the ACE I/D
polymorphism may represent a marker for another genetic polymorphism
involved in the regulation of ACE level. However, the exact identity or location of
such functional polymorphism is still unknown.
Although, the ACE I/D polymorphism influences the level of circulating ACE, no
differences in circulating ang II levels have been detected [55, 56]. However, the
pressor response to ang I [55, 57], as well as the generation of ang II after
intravenous infusion of ang I [55] has been shown to be higher in D/D carriers
compared to carriers of the I/I genotype.
The ACE I/D polymorphism has been extensively studied in relation to
cardiovascular disease (CVD). The first study reporting an association between
the ACE I/D polymorphism and CVD was published in 1992 by Cambien and
colleagues [58]. They found that the ACE D/D genotype was more frequent
among subjects who had suffered a myocardial infarction compared to healthy
subjects, especially in a subgroup of patients considered to be at low risk [58].
Some studies have confirmed an association between the D/D genotype and
myocardial infarction [59, 60], while others failed to identify any association [61,
62]. Besides myocardial infarction, the D/D genotype has been associated with
e.g. coronary artery disease [63, 64], hypertension [65, 66] and heart failure [67].
Today, there is no consensus regarding the importance of the ACE genotype for
CVD. The D/D genotype alone might not be a risk factor for CVD, but together
with other factors it may increase the risk for cardiovascular complications and
may be of particular importance in specific subgroups.
Regulation of ACE
There is a large inter-individual variation in the amount of circulating ACE, partly
due to the ACE I/D polymorphism [51]. However, ACE level is very stable when
measured in the same individual at different occasions [68]. ACE may be
regulated at the transcriptional level, affecting the synthesis of the enzyme.
Studies have shown that the expression of ACE is affected by endogenous as well
as exogenous factors e.g. vascular endothelial growth factor [69], estradiol [70],
glucocorticoids [71, 72], thyroid hormones [72], and low-salt diet [73].
Alternatively, ACE may be regulated at the protein level, changing the enzymatic
activity without altering the expression. ACE inhibitors (ACEi) are commonly
used blood pressure lowering drugs and bind to the active sites of ACE, thereby
BACKGROUND I 9
inhibiting ACE activity [74, 75]. However, a number of studies have shown that
long term treatment with ACEi induces an increased expression of ACE in
endothelial cells [76, 77], as well as in the circulation [52, 78]. Furthermore, nitric
oxide, which is produced and released from vascular endothelium, has been
shown to reduce ACE activity [79, 80], and has been suggested as an competitive
inhibitor of ACE [79]. Alternative mechanisms for regulation of ACE might involve
clearance of ACE from the circulation, as well as release and shedding of the
enzyme from ACE containing cells [45, 81].
10 I BACKGROUND
Nicotine and nicotine metabolites
Nicotine is a naturally occurring alkaloid found in many plants e.g. Nicotiana
tabacum. It is absorbed through the oral cavity, skin, lung and gastrointestinal
tract [82]. Following absorption, nicotine is metabolised to a number of
metabolites, mainly in the liver (Figure 4). As the enzymes involved in the
metabolic pathway of nicotine are highly polymorphic, there are individual
differences in the metabolic pattern of nicotine [83]. However, the pattern
appears to be consistent for in one individual over time [84].
Figure 4. Overview of the nicotine metabolism and its main metabolites.
On average, 70-80% of the nicotine is metabolised to cotinine [85], about 4% is
converted to nicotine-1´-N-oxide and 0.4% to nornicotine [85]. Cotinine is further
metabolised to cotinine-N-oxide, norcotinine and trans-3´-hydroxycotinine,
among others [84]. For most people, trans-3´-hydroxycotinine is the most
abundant metabolite in urine, accounting for on average 38% of the metabolites
[84]. The half-life of nicotine is about 2-3 hours [86] and plasma nicotine
concentration in smokers usually range between 20-40 ng/ml (0.12-0.25 µM)[84,
86]. The nicotine metabolites have considerable longer half-lives compared to
nicotine, on average 16-17 hours [85]. Due to the long elimination time, plasma
concentrations of nicotine metabolites in tobacco users tend to accumulate
BACKGROUND I 11
throughout the day [85], resulting in high levels of cotinine and trans-3´hydroxycotinine [85, 87].
Cardiovascular effects of nicotine
There is no doubt about the negative effects of smoking on the cardiovascular
system [88]. The exact mechanisms by which smoking induces CVD are not
entirely known, but are most likely multifactorial. Cigarette smoke is a complex
mixture of chemical substances, containing not only nicotine, but also a number
of other potentially cardiotoxic substances e.g. carbon monoxide [89]. Nicotine
affects cardiovascular biology in many ways, some of the mechanisms being well
characterized. By activating the sympathetic nervous system, nicotine induces
increased heart rate and myocardial contraction, vasoconstriction in the skin and
induces adrenal and neural release of catecholamines [90, 91].
In addition, animal studies have shown that nicotine affects lipid metabolism [92,
93] and accelerates the development of atherosclerosis [93, 94]. Nicotine has also
been shown to cause endothelial dysfunction [95-97], induce morphological
changes in endothelial cells [98] and increase endothelial cell death [99].
Furthermore, long-term use of oral snuff, where nicotine is absorbed through the
oral mucosa, has been shown to induce endothelial dysfunction [100] and
increase the risk of fatal myocardial infarction [101, 102]. Taken together, these
data suggest a role of nicotine in development and progression of CVD. Although,
the concentration of some of the nicotine metabolites in the blood is far higher
than nicotine in tobacco users, few earlier studies have examined their effect on
the cardiovascular system, and no previous study has examined their effect on
ACE.
12 I BACKGROUND
The abdominal aorta
The abdominal aorta is an area particularly prone to atherosclerosis as well as
aneurysm formation [103, 104]. Compared to the more proximal aorta, the
abdominal aorta contains fewer lamellar units and has a stiffer wall [105]. Most of
the abdominal aortic media lacks vasa vasorum and is instead supplied with
oxygen and nutrients by diffusion from the bloodstream [105]. Increased wall
thickness, as a result of e.g. ageing [106, 107], may therefore result in impaired
nutrition of the abdominal aortic wall. Furthermore, age- and gender-related
changes are more pronounced at this site than in other arteries [107, 108]. In
addition, the abdominal aorta is exposed to higher systolic and pulse pressures
than other central arteries [109].
Abdominal aortic aneurysm (AAA) is defined as a 50% enlargement of the aortic
diameter or a localized dilatation [110]. However, in clinical routine a diameter of
3 cm or greater is usually defined as AAA. Risk factors for AAA are e.g. old age,
male gender, smoking and hypertension. The prevalence of AAA is low in young
populations, but increases with age, reaching 5-8% in men, and 1-2% in women
at the age of 65 or above [111]. Rupture of an AAA is associated with a 80%
mortality rate [112]. The exact mechanisms involved in aneurysm formation are
largely unknown. However, a genetic component has been suggested, as first
degree relatives to patients with AAA are more susceptible. Furthermore,
previous studies have indicated a possible connection between arterial stiffness
and aortic aneurysm [113, 114].
BACKGROUND I 13
Arterial stiffness
Large artery stiffening is an independent risk factor for cardiovascular morbidity
and mortality [115, 116]. Stiffness of large arteries is determined by the content
and composition of the arterial wall. Elastin and collagen are the main structural
proteins responsible for the elastic properties of large arteries [117]. Elastin is
responsible for the mechanical strength at low pressures, while collagen is the
main load bearing protein at high pressures [118]. The elasticity varies along the
arterial tree; the thoracic aorta being the most elastic area while more distal
arteries become gradually stiffer [104, 119], as a result of reduced elastin and
increased collagen content [120]. Stiffness of the arterial wall is influenced by
age, gender and other recognised cardiovascular risk factors [121-124]. As a
consequence of ageing, the structure of the arterial wall is altered, with loss of
elastin fibres and increase in collagen content [125]. Men have stiffer arteries
than women, at least when measured locally [126-128], although ageing is
associated with increased stiffness in both men and women [128, 129]. Ageing is
also associated with an increased arterial diameter [113, 130] and thickening of
the arterial wall [130]. These age-related changes occur predominantly in elastic
arteries, while muscular arteries are less affected [121, 129]. Apart from the
classic cardiovascular risk factors, genetic factors have been implicated as
important determinants of arterial stiffness, either directly by inducing structural
changes in the vessel wall, or indirectly acting through other risk factors [131].
Previously, genetic polymorphisms in e.g. the fibrillin-1 gene [132, 133], MMP
genes [134, 135] and a region on chromosome 9p21.3 [136] has been associated
with altered arterial stiffness.
14 I BACKGROUND
Arterial pulse wave and wave reflection
The blood flow in the aorta and other large arteries is pulsatile as a result of the
rhythmic ejection of blood from the left ventricle. The left ventricular (LV)
ejection generates a pressure wave which travels along the arterial tree with a
pulse wave velocity (PWV) of 4-10 m/sec [119]. This pressure wave is reflected
at sites of changing impedance e.g. branching points, areas of alterations in
arterial elasticity or diameter, and high resistant arterioles, augmenting the
forward wave as it moves along the arterial tree [137]. The arterial pressure
wave thus represents the sum of the forward and the reflected pulse waves
(Figure 5).
Measured
Reflected
Forward
Figure 5. Schematic illustration of the aortic pressure wave in an elderly
subject.
The shape of the arterial pressure wave is determined to a large extent by arterial
stiffness. In the aorta, with low PVW, the reflecting pulse wave predominantly
returns in the diastolic phase of the forward pressure wave, adding to the
diastolic pressure resulting in low pulse pressure. With increasing age however,
increased stiffness results in premature return of the reflecting wave, adding to
the systolic pressure wave [137]. This pressure boost, caused by wave reflection
is called augmentation pressure, and generates increased systolic and pulse
pressure [137]. Due to increased afterload, a rise in systolic and pulse pressure
results in increased cardiac oxygen consumption [119, 137]. In addition, when
the reflecting waves arrive earlier in systole, the diastolic pressure decreases,
thereby reducing coronary perfusion [119, 137].
The pressure wave is progressively amplified as it moves from central (elastic) to
more distal (muscular) arteries [138]. This amplification phenomenon is due to
increased stiffness along the arterial tree and a decreased distance to reflection
sites, resulting in augmentation of the systolic pressure [119]. In healthy young
BACKGROUND I 15
subjects with elastic arteries, the pressure in peripheral arteries is thus usually
higher than in central arteries [139]. This difference decreases to a large extent
with age [137, 138], as the stiffness of muscular arteries is less affected by aging
[129]. In recent years, there has been a growing interest in studying blood
pressure in central arteries as this is the pressure the heart actually meets, and as
central pressure has been shown to be a better predictor of cardiovascular
outcome than peripheral pressure [139, 140].
Heart failure and RAS
Heart failure is a growing health problem in Western countries. The prevalence of
heart failure is low in young populations but increases with age, reaching
approximately 10% in those aged ≥65 years [141]. In systolic heart failure, the
ventricular contraction is impaired, and in the majority of patients with systolic
heart failure, the left ventricle is affected. Major determinants of systolic heart
failure are ischemic heart disease and hypertension, but a variety of other cardiac
disorders (e.g. dilated cardiomyopathy, heart valve diseases, arrhythmias) may
also be the underlying cause [142].
A chronically elevated blood pressure often results in left ventricular
hypertrophy (LVH), which represents a strong predictor of cardiovascular
morbidity and mortality [143]. Although, hemodynamic factors are considered as
main determinants of LVH [144], non-hemodynamic factors may also be of
importance [19, 145, 146]. RAS has been recognized as an important system in
the pathogenesis of heart failure. Most evidence supporting a role of RAS in heart
failure arise from the beneficial effects seen after pharmacological inhibition.
ACEi are first line therapy for patients with heart failure, and has been shown to
reduce cardiovascular morbidity and mortality in a number of clinical trials [147,
148]. In recent years, accumulating evidence indicates that these beneficial
effects are shared by angiotensin II receptor blockers (ARBs) [149, 150].
16 I BACKGROUND
AIMS
The aims of this thesis were to …
Study the effect of nicotine and nicotine metabolites on ACE in cultured
endothelial cells (HUVECs) and to evaluate the possible influence of the ACE I/D
polymorphism (paper I).
Study the association between the ACE I/D polymorphism, circulating ACE level
and cardiovascular risk in elderly men and women (paper II).
Study the association of the ACE I/D polymorphism, circulating ACE and the
mechanical properties of the abdominal aorta in elderly men and women (paper
III).
Study the association of the ACE I/D polymorphism and circulating ACE levels,
with central aortic blood pressure, aortic augmentation, pulse pressure
amplification and LV function in elderly subjects (paper IV).
AIMS I 17
MATERIALS AND METHODS
Cell culturing, HUVECs (paper I)
The cultured cells used in paper I, were Human Umbilical Vein Endothelial Cells
(HUVECs) isolated from umbilical cords after vaginal deliveries without
complications. Umbilical cords are suitable for isolation of endothelial cells as
they are easy to obtain and have an unbranched vein of appropriate size. The
method for isolation and cultivation of HUVECs was first described by Jaffe et al.
in 1973 [151].
Umbilical veins were treated with collagenase for detachment of endothelial cells
from the vessel. The collagenase-cell suspension were collected and HUVECs
were seeded in cell culture flasks coated with 0.2% gelatin. Endothelial cells
grown on uncoated plastic have low spontaneous proliferation and relatively high
apoptosis, whereas growth on surfaces of e.g. gelatin is more optimal [152]. Cell
culture medium was replaced every 48-72 h and confluent cells were trypsinised
and subcultured in new cell culture flasks. As HUVECs are primary culture cells
they have limited life span and culturing for more than 2-3 passages results in
reduced proliferation rate. In addition, the expression of ACE in HUVECs is
reduced by cultivation [153]. HUVECs were thus used at the first or second
passage.
MATERIALS & METHODS I 19
Study populations (paper II-IV)
In paper II-IV we studied elderly subjects from a rural municipality in South East
of Sweden. All subjects were included in a previous study which started in 1999
[154]. When a follow-up study was performed between 2003 and 2005 we had
the opportunity to perform a cross-sectional study in this population. A total of
672 subjects (322 men and 350 women) were included (paper II), of whom 655
subjects had successful examination of their LV function (paper IV). All 672
subjects were also asked to participate in a study regarding mechanical
properties of the abdominal aorta as well as central hemodynamics. A total of 452
agreed, resulting in a participation rate of 67%. As the study population consisted
of elderly subject, and many of them had quite a long distance to the clinic, the
main reason for not participating was transportation problems. Non-participants
were slightly older compared to those who chose to participate, however there
were no difference in blood pressure or CVD. In addition, there were more men
than women who agreed to take part. Determination of abdominal aortic wall
mechanics was successful in 406 subjects (paper III), while central aortic
hemodynamics were successfully obtained in 422 subjects (paper IV). Figure 6
shows the study populations and the examinations that were performed.
672 subjects
Paper II
Paper IV
ACE genotype and ACE level
452 subjects
655 subjects
Echocardiography
Examination of abdominal aorta and
measurement of central aortic hemodynamics
406 subjects
422 subjects
Mechanical properties of
abdominal aorta
Central aortic hemodynamics
Paper III
Paper IV
Figure 6. Study populations used in paper II-IV
20 I MATERIALS & METHODS
Determination of ACE activity
ACE activity was determined in cultured endothelial cells (paper I) and in serum
(paper I-II), using a commercially available radio-enzymatic assay (ACE-direct
REA, Bühlmann Laboratories, Schönenbuch, Switzerland). The principle for the
assay is based on the cleavage of the synthetic substrate 3H-hippuryl-glycylglycine into 3H-hippuric acid and glycyl-glycine dipeptide, a reaction catalyzed by
ACE. Adding of HCl stops the enzymatic reaction and the scintillation cocktail
separates the 3H-hippuric acid from the glycyl-glycine dipeptide. The amount of 3Hhippuric acid can be determined using a beta counter, and is proportional to the
amount of ACE in the sample. One unit of ACE is defined as the amount of enzyme
required to produce 1 µmol 3H-hippuric acid per minute and litre. Intra-assay
variation was 8% for ACE activity determined in cultured cells, and 6% in serum,
while inter-assay variation was 7%.
Determination of ACE level
ACE level was measured in cell lysate (paper I), in serum (paper I and II) and in
plasma (paper II-IV), using Enzyme-linked immunosorbent assay (ELISA)
(Quantikine, Human ACE Immunoassay, R&D Systems, Minneapolis, USA). The
principle for the assay is as follows; monoclonal antibodies specific for ACE are
coated on the bottom of a 96-well microplate. Samples are added to the wells and
any ACE present in the samples bind to the antibodies. Unbound substances are
removed by washing. Biotinylated polyclonal antibodies directed against ACE are
added, followed by addition of streptavidine-horseradish peroxidase, attaching to
the polyclonal antibody. A substrate is then added, which is converted to a
coloured compound by horseradish peroxidase. The intensity of the colour is
measured spectrophotometrically and is proportional to the amount of ACE in the
sample. Standards with known concentration of ACE were included in each assay
and used to calculate the concentration of ACE in samples. Serum and plasma
samples were diluted 1:200. Samples were analysed in duplicate and re-analysed
if variation from the mean value exceeded 15%. The lower limit of detection was
0.05 ng/ml. Intra-assay variation was 6.5% for analysis of ACE in plasma, and 8%
in cell lysate, while inter-assay variation was 12%.
MATERIALS & METHODS I 21
Validation of ACE assays
Although a good correlation between ACE activity and ACE level in plasma has
been shown previously [155], we wanted to confirm this using our methods in a
population similar to our study population. Serum and plasma were collected
from 23 men, 70 years of age, enrolled in a screening program for abdominal
aortic aneurysm at Linköping University Hospital. These subjects represent an
unselected sample of a normal population of elderly men in Sweden. ACE activity
was measured in serum, while ACE level was measured in serum and plasma,
according to the methods described above. There was a strong correlation
between ACE activity and ACE level in serum (Figure 7A) as well as in serumplasma (Figure 7B), indicating that ACE level can be used as a surrogate for ACE
activity. A similar correlation was also found in a younger healthy population
including both men and women (12 men and 9 women, mean age 29 years)
(unpublished data).
A
B
Figure 7. Correlation between A) ACE level and ACE activity in serum, and B)
between ACE level in plasma and ACE activity in serum, from 23 men, 70 years old.
22 I MATERIALS & METHODS
Stability of ACE during storage at -70°C
In paper II-IV, ACE level was analysed in plasma stored at -70°C for 3-5 years. As
freezing might result in degradation of proteins the stability of ACE in frozen
samples was studied. Plasma and serum from 23 men enrolled in a screening
program for abdominal aortic aneurysm at Linköping University Hospital was
used (see above). The method for preparation of plasma and serum has been
described in paper II. Samples were aliquoted and stored in 1.5 ml cryo-tubes at
-70°C and analysis of ACE level was planned after 3 weeks, 12, 24 and 48 months.
To date, samples stored at -70°C for 3 weeks and 12 months respectively, have
been analysed.
As shown in Figure 8, there was no difference in ACE level between the 3 week
and the 12 months samples, indicating that there is no degradation of ACE at least
during the first year of freezing.
Figure 8. ACE level (ng/ml) in plasma samples from 23 men after 3 weeks and
12 months of storage at -70°C. Values are mean ± SEM.
MATERIALS & METHODS I 23
ACE genotyping
A polymerase chain reaction (PCR) method for determination of ACE genotype
was described by Rigat and colleagues in 1992 [156]. A few years later, this
method was found to result in mistyping of a number of subjects, where carriers
of the I/D genotype were incorrectly identified as D/D carriers [157]. More
recently, improved methods for ACE genotyping have been described [158-160].
We used a triple primer approach [158], a strategy previously described to be
reliable to avoid mistyping of I/D carriers [159]. Three sets of primers were used,
allowing detection of a 238 bp fragment for the deletion allele, and two
fragments, 155 bp and 525 bp for the insertion allele [158] (Figure 9).
Deletion allele
primer
primer
DNA
238 bp PCR-product
Insertion allele
primer
I-allele specific primer
primer
DNA
155 bp PCR-product
525 bp PCR-product
Figure 9. Schematic illustration of the method used for ACE genotyping. The forward
primer (red) and the reverse primer (blue) allow detection of a 238 bp fragment for
the D allele, and 525 bp fragment for the I-allele. By using an I-allele specific primer
(green), an additional fragment is amplified for the I-allele, making determination of
ACE genotype easier and more accurate.
Amplified DNA was separated by gel electrophoresis using a 1.5% agarose gel
stained with ethidium bromide and visualized by UV-light. Determination of ACE
genotype was based on length and number of DNA fragments (Figure 10).
DD
II
ID
525 bp
238 bp
155 bp
24 I MATERIALS & METHODS
Figure 10. PCR products separated by gelelectrophoresis
and visualised by UV-light. The D/D genotype results in
one band, I/I genotype two bands and I/D genotype
three bands.
Determination of abdominal aortic wall mechanics
Stiffness of various arteries can be determined non-invasively by a number of
different techniques [119]. Carotid-femoral PWV measurements are considered
the golden standard for measurement of aortic stiffness [119]. However, as PWV
usually is measured between the carotid and femoral arteries, it reflects the mean
arterial stiffness of several different territories of the arterial tree. In paper III, we
used ultrasound and the Wall Track System, as we wanted to determine arterial
stiffness locally in the abdominal aorta. This technique enables high resolution
measurements of lumen diameter, pulsatile diameter changes during a cardiac
cycle, and intima-media thickness (IMT) [161], which can be used to calculate
local aortic stiffness. The aorta was examined approximately 3-4 cm proximal to
the aortic bifurcation, a site particularly prone to aneurysm formation [104]. ECG
electrodes were connected to the subject. The abdominal aorta was visualized
longitudinally and the scanner was then switched to M-mode. The Wall Track
System automatically positions two anchors at the posterior and the anterior
aortic wall. Manual adjustments of the anchor positions can be made. Pulsatile
vessel wall movements were recorded and arterial distension waveforms
generated (Figure 11), allowing calculation of lumen diameter and pulsatile
diameter changes. IMT of the posterior wall was measured in end-diastole, using
the ECG signal for calibration. The Wall Track System uses the radio frequency to
automatically determine IMT from the interface between the lumen and the
intima, to the interface between the media and adventitia [161]. Each
measurement was evaluated and compared with the ultrasonic image. Only
measurements which were in agreement with the visual estimation were
included. All measurements were carried out by two experienced
ultrasonographers on a single occasion, with subjects in the supine position,
immediately following brachial blood pressure measurements. Coefficient of
variation was 5% for absolute lumen diameter, 21% for pulsatile diameter
change and 17% for IMT.
Figure 11. Distention curves from the abdominal aorta determined by the Wall
Track System.
MATERIALS & METHODS I 25
Using diastolic lumen diameter, pulsatile diameter change and brachial blood
pressure, the compliance coefficient (CC) and distensibility coefficient (DC) were
calculated using the formulae [129]:
(
)
CC = π 2 × Ddia × ∆D + ∆D 2 / (4 × ∆P )
(
)(
2
DC = 2 × Ddia × ∆D + ∆D 2 / Ddia × ∆P
)
CC is expressed in mm2/kPa and DC in 10-3/kPa. Ddia is the end diastolic diameter
(mm), ΔD is the diameter change between systole and diastole (mm), and ΔP is
the brachial pulse pressure expressed in kPa.
CC is the absolute change in cross-sectional area during a cardiac cycle for a given
increase in aortic pressure, assuming that the length of the vessel is constant
during the pulse wave. A low CC indicates reduced vessel buffering capacity. DC is
the relative change in aortic diameter during a cardiac cycle for a given increase
in pressure. A decreased DC indicates reduced elasticity of the vessel. There is a
non-linear relationship between pressure and diameter change in the abdominal
aorta [162, 163]. The aorta is very distensible at low pressures and small
diameters, but becomes gradually stiffer with increasing pressure and diameter
[162, 163]. Stiffness β is an index which seems to be less dependent on pressure
changes [162], and may be used as a complement to CC and DC. Stiffness β was
calculated according to [162, 164]:
Stiffnessβ = ln (Psys / Pdia ) / (∆D / Ddia )
Psys and Pdia represent the systolic and diastolic brachial blood pressures in
mmHg. Stiffness β varies inversely with DC and CC.
Brachial blood pressure, determined using an oscillometric technique (Dinamap
model PRO 200 Monitor, Critikon, Tampa, FL, USA) was used as a surrogate for
abdominal aortic pressure, in order to calculate CC, DC and stiffness β.
Simultaneous measurement of aortic pressure would be ideal, but is difficult to
perform and unethical in large populations of elderly subjects. The systolic
pressure in the brachial artery and in the abdominal aorta are in good agreement
[163]. Diastolic pressure on the other hand, is slightly higher in the brachial
artery, leading to a systematic underestimation of aortic stiffness [163]. However,
as no age- or gender-related differences have been observed [163], this
systematic bias should not affect comparative studies between groups.
26 I MATERIALS & METHODS
Central aortic hemodynamics
There is a growing interest in studying blood pressure in central arteries, as this
is the pressure the heart actually meets. Invasive measurement is the most
accurate assessment of central aortic pressure, however this is not very ethical in
large populations of elderly subjects. Instead, two non-invasive techniques are
commonly used; direct estimations of central aortic pressure from the carotid
pressure wave form, or calculation of central aortic pressure using tonometric
recordings of the radial pressure waveform and a generalized transfer function.
In contrast to the carotid artery, the radial artery is supported by bone structures,
making it easier to obtain high quality pressure curves [119]. In Paper IV, central
aortic pressure was determined from the radial artery. A Millar pressure
tonometer was placed on the radial artery and pressure waveforms were
recorded. Using brachial blood pressure for calibration, the SphygmoCor system
was used to synthesize aortic pressure waveforms (Figure 12).
Aortic
Radial
Psys
Aug
PP
Pdia
Figure 12. Radial and aortic pressure waveforms obtained by applanation tonometry
and the SphygmoCor system. Psys: systolic pressure, Pdia: diastolic pressure, Aug:
augmentation pressure, PP: Pulse pressure.
This method has been validated and showed good agreement with invasively
determined pressure, when using intra-radial pressure for calibration [165, 166].
Using brachial blood pressure for calibration, underestimation of central aortic
pulse pressure and systolic pressure has been reported [167, 168]. However, this
bias is probably less pronounced in elderly populations like ours, as the pressure
MATERIALS & METHODS I 27
differences in the arterial tree decrease with age [125]. Moreover, a systematic
underestimation of central blood pressure should not affect comparative studies
between groups.
From the central aortic pressure waveform a number of hemodynamic
parameters can be obtained. Pulse pressure is the difference between the systolic
and diastolic pressure. Augmentation pressure is the difference between the first
and the second systolic peak and represents the pressure boost caused by wave
reflection. Augmentation index (AIx) is the augmentation pressure expressed as
percentage of the aortic pulse pressure, and is considered as an indirect measure
of aortic stiffness. AIx is dependent on heart rate and AIx is therefore adjusted to
a heart rate of 75 beats/minute (AIx HR75). Time to reflection represents the
time to return of the reflected wave.
Echocardiographic evaluation of left ventricular function
In paper IV, LV function was determined semi-quantitatively by visual estimation
using doppler echocardiography (Acuson XP 128c system, Mountain View, CA,
USA), a method regularly used in both clinical routine and research at our
department. The method has been shown to be in good agreement with Simpsons
biplane method as well as radionuclide imaging [169-171]. A good correlation
between visual estimation and Simpsons biplane method has also been confirmed
in our lab [172]. Subjects were classified into four groups based on LV function;
normal function, mild dysfunction, moderate dysfunction or severe dysfunction,
corresponding to LV ejection fraction of >50%, 40-49%, 30-39%, <30 %
respectively. Examinations were performed by three experienced physiciansechocardiographers with the subjects in the left supine position.
28 I MATERIALS & METHODS
RESULTS AND DISCUSSION
Effect of nicotine and nicotine metabolites on ACE in vitro
Cardiovascular effects of nicotine have been extensively studied, and it seems as
nicotine can promote development of atherosclerosis [93-96]. The impact of
nicotine metabolites on the cardiovascular system is however rather unknown,
despite high plasma concentrations of some of the metabolites in daily smokers.
In paper I, the effect of nicotine as well as nicotine metabolites on ACE in human
endothelial cells and in human serum was studied. Nicotine, and the five most
abundant metabolites found in plasma from tobacco users were used; cotinine,
cotinine-N-oxide, nicotine-1´-N-oxide, norcotinine, and trans-3´-hydroxycotinine
in concentrations similar to those observed in plasma from daily smokers [8487].
Nicotine, as well as some of the nicotine metabolites induced an increase in ACE
activity in HUVECs (Figure 13). The effect was dose-dependent, except for
nicotine 10 µM. The effect of nicotine and nicotine metabolites on ACE activity
was studied after 10 min-24h incubation. A slight increase in ACE activity was
seen after 10 min, which in some cases sustained up to 24 h (data not shown).
The most pronounced effect was however seen after 30-60 min, and a 60 min
incubation time was therefore chosen for subsequent experiments.
RESULTS & DISCUSSION I 29
A
Nicotine
ACE activity (U)
50
*
40
30
20
20
10
10
0
0
C
Norcotinine
ACE activity (U)
** **
40
D Trans-3-hydroxycotinine
40
30
20
20
10
10
0
0
E
Nicotine-1'-N-oxide
*
50
30
50
Cotinine-N-oxide
40
30
50
ACE activity (U)
**
B
50
F
50
40
40
30
30
20
20
10
10
0
0
*
** **
Cotinine
Figure 13. Effect of nicotine and nicotine metabolites on ACE activity in HUVECs.
ACE activity was measured in HUVECs following incubation with A) nicotine,
B) cotinine-N-oxide, C) norcotinine, D) trans-3-hydroxycotinine, E) nicotine-1´-Noxide and F) cotinine for 1 h. Values are mean ± SEM. n = 11-16
30 I RESULTS & DISCUSSION
The expression of ACE was slightly increased by nicotine, cotinine-N-oxide and
nicotine-1’N-oxide, although no dose-dependent effects were seen (Figure 3,
paper I). Pre-treatment of HUVECs with the protein synthesis inhibitor
cycloheximide, followed by incubation with nicotine showed that nicotine was
still able to induce an elevation in ACE activity, although the effect was slightly
reduced at the highest concentrations of nicotine (Figure 14). If the increase in
ACE activity was entirely due to increased expression of the enzyme, incubation
with a protein synthesis inhibitor would abolish the effect. This suggests that the
increase in ACE activity after treatment with nicotine and nicotine metabolites is
mainly due to altered enzymatic activity, but also due to a slight increase in the
expression, at least at high concentrations.
ACE activity (U)
30
Figure 14. Effect of nicotine on ACE
activity after pre-incubation with
cycloheximide (10µM). Despite pretreatment with a protein synthesis
inhibitor, nicotine induced an
increased ACE activity, although the
effect was slightly reduced at 1 and
10 µM.
28
26
24
22
20
0 µM
nicotine
0.1 µM
1 µM
10 µM
cykloheximide + nicotine
To further evaluate the mechanism of ACE regulation, ACE activity was analyzed
in serum from 3 healthy non-tobacco using volunteers after incubation with
nicotine (Figure 5, paper I). There was no effect of nicotine on serum ACE,
indicating that the regulation of ACE by nicotine only takes place within
cells/tissues.
Previous studies investigating the effect of nicotine on ACE have produced
conflicting results. Saijonmaa and colleagues [69] found no effects of nicotine
alone after 4, 18 or 24 hours incubation, but together with vascular endothelial
growth factor (VEGF), nicotine potentiated the VEGF-induced up-regulation of
ACE in HUVECs. The difference between our study and the study by Saijonmaa et
al. may be related to methodological differences as well as the time of incubation.
Zhang and co-workers [173], on the other hand, showed an up-regulation of ACE
RESULTS & DISCUSSION I 31
mRNA in cultured human coronary artery endothelial cells after 24 hours
incubation with nicotine, and Sugiyama et al. [174] showed increased serum ACE
activity in dogs 30-60 min after intravenous administration of nicotine. Although
the effect of nicotine was studied at different time points, and despite differences
in methods as well as cells/animal models, the data by Zhang et al. and Sugiyama
et al. are in agreement with our findings, and supports a nicotine-induced
increase in ACE activity. Interestingly, a protective effect of the ACEi captopril on
nicotine-induced endothelial dysfunction has been shown in vitro and in vivo
[175], suggesting that nicotine may promote atherosclerosis by inducing an upregulation of ACE. Our data also suggests a similar effect of some of the nicotine
metabolites (i.e. norcotinine, trans-3´-hydroxycotinine and cotinine-N-oxide). Due
to high concentrations of nicotine metabolites in plasma, it is of great importance
that the effect of the metabolites is considered.
Individual differences in the response to nicotine and nicotine metabolites
Although, mean values showed an increased ACE activity following treatment
with nicotine or nicotine metabolites, there were individual differences in the
response; some experiments showed a 50% increase, whereas in others no or a
moderate effect was seen (Figure 15).
I
60
II
45
30
III
10 µM
1 µM
0
0.1 µM
15
0 µM
ACE activity (U)
75
Figure 15. Illustration of individual differences in ACE activity in HUVECs
from three umbilical cords, after treatment with trans-3´-hydroxycotinine.
32 I RESULTS & DISCUSSION
This difference was not related to either basal ACE activity or time of culture. In
each separate experiment, HUVECs obtained from different umbilical cords with
individual genetic background, were used. As the ACE I/D polymorphism is
known to influence ACE level [51] and as previous studies have reported that the
effects of e.g. nitric oxide and ACEi, are dependent on ACE genotype [80, 176], we
hypothesized that the variation in response to nicotine and nicotine metabolites
seen in our study might be related to the ACE genotype. However, our data could
not confirm such interaction, and the explanation for this variation remains
unknown. One might speculate that life style factors (e.g. tobacco or diet) would
explain differences in response to nicotine and nicotine metabolites. However, as
the donors of the umbilical cords were anonymous, no such information was
available leaving this relationship to be further elucidated. If this individual
difference also applies in vivo, it may be of importance regarding risk for CVD in
tobacco users, where some subjects might be more affected.
RESULTS & DISCUSSION I 33
Association between ACE and cardiovascular risk
Numerous studies have investigated the link between the ACE I/D polymorphism
and CVD. Although a relationship between the ACE I/D polymorphism and
plasma ACE level has been repeatedly confirmed [51, 52], other factors have been
shown to influence circulating ACE levels [70, 73, 177-180]. Despite this, few
studies have investigated the role of circulating ACE in CVD. In addition to
elucidate the relationship between ACE genotype and CVD, this study also focuses
on the link between ACE level, cardiovascular risk factors and CVD.
ACE level – influence of ACE genotype, age and gender
A study by Rigat et al. [51] including 80 healthy middle aged men and women
showed that 47% of the variation in circulating ACE was due to the ACE I/D
polymorphism. Subsequent studies in larger populations have confirmed the
impact of the ACE I/D polymorphism on circulating ACE, but have showed a less
pronounced effect, accounting for approximately 20% of the variation [52]. Our
study showed a significant association between the ACE I/D polymorphism and
circulating ACE, although there were large variations within each group (Figure
16). Only 10% of the variation in men and 17% in women were due to the ACE
I/D polymorphism.
Figure 16. Impact of the ACE I/D polymorphism on circulating ACE level in
elderly men and women.
34 I RESULTS & DISCUSSION
There were no gender differences in ACE level in the overall population or
between genotypes. In previous studies, gender differences have been reported
among children, boys having higher circulating levels of ACE than girls of same
age [181, 182]. However, this gender-related difference seems to diminish with
age, as most investigations in adults failed to identify such association [51, 183].
The frequency of the D allele in our population was 0.51, which is in accordance
with previous studies in Caucasian populations [61, 184]. However, ethnical
differences in allele frequency [185-187], as well as the impact of ACE I/D
polymorphism on circulating ACE have been shown [181, 188]. Furthermore,
there was a positive association between age and ACE level in our study. A
negative association between age and ACE level has been shown in children [181,
189], however, studies in adults showed no influence of age on ACE level [68, 190,
191]. Whether the age related increase in ACE seen in our population is due to
higher prevalence of diseases in older subjects, or to age per se remains
unknown. However, age obviously needs to be taken into consideration when
studying circulating ACE levels, at least in elderly subjects.
ACE level and cardiovascular risk factors
To examine the relationship between ACE level and cardiovascular risk, subjects
were divided into 4 groups based on the number of cardiovascular risk factors.
Risk factors were diabetes, heredity for CVD, hyperlipidemia and hypertension. In
men, there was a positive association between number of risk factor and ACE,
while no association was seen in women (Figure 17).
Figure 17. Circulating ACE levels in men and women according to number of
cardiovascular risk factors. P-values are ONE-way ANOVA adjusted for age and ACE
genotype.
RESULTS & DISCUSSION I 35
Elevated ACE levels may represent one of the cellular mechanism involved in the
vascular damage associated with cardiovascular risk factors, possibly mediated
by increased production of ang II, increased degradation of bradykinin and
altered levels of other angiotensin peptides e.g. angiotensin 1-7. To further
investigate which particular risk factors that are associated with higher ACE
levels in men a multivariable analysis including risk factors and CVD was
performed.
Higher ACE levels were seen in men who smoked compared to non-smoking men,
while no difference was found in women. However, no interaction between
gender and smoking was seen. The lack of association in women may be due to
the low proportion of female smokers. An increased release of ACE from cultured
endothelial cells after exposure to cigarette smoke has been reported previously
[192]. In addition, in vivo studies have indicated that the acute effects of smoking
is an increased serum ACE activity [174, 193], whereas results regarding the
long-term effects are inconsistent [194-196]. A decreased ACE activity has been
reported in daily smokers [194, 195], however, a relatively small number of
subjects were studied and no confounders were considered. A larger study, on
the other hand, which included correction for a number of potentially
confounding factors (e.g. ACE-genotype, age, and gender) showed higher ACE
level among smokers, which is in line with our findings [196]. Taken together, our
data, as well as previous studies suggests that smoking induces an up-regulation
of circulating ACE [192, 196], possibly mediated by nicotine and nicotine
metabolites [69, 173, 197].
Furthermore, a gender-dependent association between ACE and diabetes was
found, where diabetic men had higher ACE levels compared to men without
diabetes, while no such difference was seen among women. Higher ACE levels
have previously been reported in diabetic patients compared to healthy controls
[198-200], however, no difference between genders was reported. At present, the
rationale for this gender difference is unknown and needs to be further
elucidated and confirmed in future studies. Diabetic nephropathy is a common
complication in diabetics, and previous studies have shown up-regulation of ACE
in diabetic nephropathy [198, 199, 201]. In our study, adjustment for glomerular
filtration rate did not affect the association between ACE and diabetes, suggesting
that this association is not due to impaired renal function. An up-regulation of
ACE may contribute to increased risk for vascular complications in diabetics. This
is supported by the findings that ACEi and ARBs reduces the risk for
36 I RESULTS & DISCUSSION
cardiovascular complications among diabetic subjects, more effectively than
placebo or other anti-hypertensive medications [202, 203].
ACE inhibitor treatment
Among the 672 studied subjects, 21% (n=141) were treated with ACEi. ACEi bind
to the active sites of ACE, inhibiting the conversion of ang I to ang II, as well as the
degradation of bradykinin. Our data showed that treated subjects had higher
levels of circulating ACE, compared to non-treated subjects. In treated men, ACE
level was 300 ng/ml compared to 195 ng/ml in non-treated men (p<0.001). The
corresponding levels for women were 343 ng/ml and 208 ng/ml (p<0.001),
respectively. An up-regulation of ACE after treatment with ACEi has previously
been reported in cultured endothelial cells [76, 77, 204], in different tissues in
animal models [77, 205], as well as in human plasma [52, 78, 206]. Furthermore,
we found no effect of ARBs on ACE levels, which is in accordance with previous
findings [204, 206]. Recently, Kohlstedt et al. [207] showed that binding of ACEi
to ACE induce an intracellular signal in endothelial cells leading to induction of
ACE mRNA expression. This molecular mechanism is probably responsible for the
up-regulation of ACE during ACEi treatment, and provides an explanation for the
lack of effect of ARBs. Due to interference of ACEi treatment on circulating ACE
levels, subjects who were on this treatment were excluded from statistical
analyses of ACE levels.
RESULTS & DISCUSSION I 37
Association between ACE and abdominal aortic wall mechanics
The abdominal aorta is a site particularly prone to aneurysm formation [103].
Age- and gender-related changes are more pronounced at this site compared to
other vascular territories. The mechanical properties of the abdominal aorta are
measures of aortic wall integrity. Impaired wall integrity, along with other local
hemodynamic and/or inflammatory factors may be of importance for aneurysm
formation. Previous studies have indicated that RAS might have a role in
regulation of vessel wall mechanics [208, 209] as well as in AAA [210, 211]. In our
study we investigated the link between ACE and abdominal aortic wall
mechanics.
ACE I/D polymorphism and abdominal aortic wall mechanics
Differences in abdominal aortic wall properties between ACE genotypes were
analysed in men and women. Men carrying the D allele had significantly lower
pulsatile diameter change (p=0.014) and DC (p=0.017) than men carrying the I/I
genotype (Figure 18). Carriers of the I/D and D/D genotype had similar stiffness
values, while carriers of the I/I genotype were the ones that differed.
Furthermore, in men, correction for a large number of potential confounders
(age, MAP, BMI, heart rate, LDL, C-reactive protein, diabetes mellitus, smoking,
antihypertensive treatment and ACE level) resulted in additional associations
between the ACE D allele and both reduced CC (p=0.045) and increased stiffness
β (p=0.048), and strengthening of the association between the D allele and DC
(p=0.003). In women, however, there were no differences in abdominal aortic
wall mechanics between genotypes.
Figure 18. Distensibility coefficient in men carrying the ACE D allele
compared to men carrying the I/I genotype. Data are mean values ± SEM.
38 I RESULTS & DISCUSSION
No previous study has investigated the impact of the ACE I/D polymorphism on
mechanical properties of the abdominal aorta. However, the ACE D allele has
been associated with increased stiffness of the carotid artery in a general
population [212] and in older subjects [213]. A number of studies have
investigated the connection between ACE I/D polymorphism and carotid-femoral
PWV, some showing no impact of the ACE I/D polymorphism on PWV [213-216],
while the I allele was associated with higher PWV in diabetics [215], in
hypertensives [217] and in healthy middle-aged subjects [218]. PWV is usually
measured between the carotid and femoral artery and reflects the mean arterial
stiffness of several arterial territories, including both elastic and muscular
arteries. PWV may thus reflect other aspects of vascular function than those
measured in our study. In addition, the elastic behaviour of various types of
arteries may be different, and genetic determinants may have different outcome
depending on the type of artery studied [213, 219].
Circulating ACE and arterial stiffness
The mechanism by which the ACE D allele modulates arterial stiffness is
unknown. A plausible mechanism could be an increased generation of ang II, due
to higher levels of ACE in carriers of the D allele. Ang II influences vascular
homeostasis, vascular tone, vascular smooth muscle cell growth [12, 13] and
affects the production of collagen and elastin in the vessel wall [15, 16], thereby
promoting stiffening of the artery. In addition, a role of ACE in arterial stiffening
is supported by the finding that ACEi and ARBs reduce arterial stiffness beyond
that expected from the reduction in blood pressure alone [208, 209, 220].
Although an association between the ACE I/D polymorphism and circulating ACE
levels has been confirmed repeatedly, we and others have shown large variations
between carriers of the same genotype [52]. Thus, we hypothesized that ACE
level would show a stronger association to arterial stiffness than the ACE I/D
polymorphism. Surprisingly, although the D allele was associated with abdominal
aortic stiffness, there was no link between circulating ACE and aortic stiffness. To
the best of our knowledge, no previous study has investigated the association
between ACE level and arterial stiffness. We do not know whether levels of
circulating ACE correlate with the levels in the arterial wall. Thus, there might be
a link between ACE in the vessel wall and abdominal aortic stiffness. The
beneficial effect of ACEi and ARBs on arterial stiffness may thus result from local
effects within the vessels wall. On the other hand, the ACE D allele may be in
linkage disequilibrium with genetic variations in other genes, which in turn may
influence aortic stiffness. The ACE I/D polymorphism may thus represent a
RESULTS & DISCUSSION I 39
marker for another genetic polymorphism involved in regulation of vessel wall
mechanics. The identity of such polymorphism is however unknown.
ACE, abdominal aortic stiffness and aneurysm
The abdominal aorta is the most common site for aneurysm formation [103]. The
mechanisms involved in aneurysm formation are largely unknown, however it
has been argued that a combination of local hemodynamic factors, as well as
factors affecting wall strength (genetic factors, proteolytic activity, inflammation
etc.) are of importance [103].
The prevalence of AAA in our population was 8% in men and 1% in women,
which is similar to previous reports [111]. There was a tendency towards higher
stiffness values in men with AAA compared to those without, however, this failed
to reach statistical significance, possibly due to the low number of subjects with
AAA. Increased abdominal aortic stiffness in subjects with AAA has on the other
hand been shown previously [113, 114].
A link between RAS and abdominal aortic aneurysm has been shown in
experimental studies [210, 211]. Accumulation of ACE has been reported in the
abdominal aortic wall in patients with aneurysm [210], and ang II infusion
induces AAA in mice [211]. Furthermore, previous studies have indicated a
possible association between the ACE I/D polymorphism and AAA [221-224].
Interestingly, Jones et al. reported that the increased risk for AAA was equal for
I/D and D/D carriers, when compared to I/I carriers [223]. The same pattern was
shown in our study; increased stiffness in carriers of the D allele independent of
one or two copies of the allele.
Our study showed an association between the ACE D allele and aortic stiffness in
men, whereas no association was found in women. This gender difference is
particularly interesting in the context of aneurysm formation, as AAA is more
common in men [103]. Previous studies investigating the link between the ACE
I/D polymorphism and arterial stiffness of other vascular territories have
analyzed mixed populations [212, 213, 216, 217]. The lack of association between
the ACE D allele and aortic stiffness in women may therefore conceal the
association in men, if mixed populations are studied.
Taken together, this study provided evidence of an association between the ACE
D allele and aortic stiffness in men. An increased abdominal aortic stiffness
indicates impaired vessel wall integrity, which may be of importance in aneurysm
40 I RESULTS & DISCUSSION
formation. However, as the ACE D allele is a common allele, it cannot be the sole
factor responsible, but together with other local factors, the ACE D allele may
predispose to aneurysm formation. An illustration of this hypothesis is shown in
Figure 19.
ACE D allele
Male gender
Loss of vessel wall
integrity in the
abdominal aorta
Local hemodynamic
and/or inflammatory
factors
Abdominal
aortic aneurysm
Figure 19. Schematic illustration of a suggested link between the ACE D allele,
abdominal aortic wall integrity and abdominal aortic aneurysm.
RESULTS & DISCUSSION I 41
Association between ACE, left ventricular function and central
hemodynamics
LV dysfunction is a common final outcome for a number of cardiac disorders.
Ischemic heart disease and hypertension are important determinants of LV
dysfunction, but a variety of other cardiac disorders (e.g. dilated cardiomyopathy,
heart valve diseases, arrhythmias) may also be the underlying cause [142].
Pharmacological inhibition of ACE is the standard treatment for patients with
heart failure and has been shown to reduce morbidity and mortality [147]. A
large meta-analysis of previously published trials demonstrated a significant
improvement in LV function in subjects treated with ACEi compared to placebo
[225]. In our study, the relation between ACE, LV function and central
hemodynamics was investigated in a cohort of elderly men and women. Subjects
with LV dysfunction had higher levels of circulating ACE than subjects with
normal LV function, and there seem to be a relationship between ACE level and
degree of LV dysfunction (Figure 20).
Figure 20. Association between circulating ACE level and LV dysfunction in elderly
subjects. Values are mean ± SEM. P-value was calculated using ONE-way ANOVA.
42 I RESULTS & DISCUSSION
The underlying cause for LV dysfunction is not known for all subjects included,
and the rationale for higher ACE levels among those with LV dysfunction is
unknown. Previous data have indicated a role of ACE in both cardiac remodeling
and ischemic heart disease. A role of ACE in LV remodeling is supported by
studies in rats, showing increased conversion of ang I to ang II as well as
increased expression of ACE in hypertrophic myocardium [226-228]. In humans,
a positive association between circulating ACE and LV wall thickness [229, 230]
and LV mass index [229, 231] has been reported. However, in contrast to our
findings, no correlation between ACE and LV function was found [230]. An
increased expression of ACE may be involved in cardiac remodelling through an
increased production of ang II and degradation of bradykinin. In the heart, ang II
has hypertrophic effects [19] and promotes fibrosis [20]. An increased
degradation of bradykinin may also promote cardiac hypertrophy, as a result of a
reduced production of nitric oxide and prostacyclin. On the other hand, no
difference in circulating ACE between normotensive and hypertensive subjects
with or without LV hypertrophy was found [145] and no relation between
circulating ACE and LV mass was detected in healthy normotensive men [183].
Furthermore, accumulation of ACE has been reported in coronary atherosclerotic
lesions [42, 232], and enhanced cardiac ACE [233], as well as circulating ACE
levels [52, 234] has been shown in patients who have suffered a myocardial
infarction. In addition, in subjects with ischemic heart disease a higher cardiac
ACE was associated with reduced LV function [235], and up-regulation of cardiac
ACE has been shown in patients with heart failure [228, 236].
In recent years, there has been a growing interest in studying blood pressure in
central arteries, as this is the pressure the heart actually meets. In addition
central pressure is more strongly related to cardiovascular outcome than the
peripheral pressure [139, 140]. Previous studies have indicated that ACEi lower
central blood pressure more effectively than other hypertensive drugs [237, 238],
suggesting a role of ACE in regulation of central hemodynamics. Ang II, the main
effector peptide of ACE, is a powerful vasoconstrictor with well-described effects
on blood pressure. As ang II raises blood pressure, one might expect to find a
connection between ACE and blood pressure. Previous data have indicated a
weak but significant association between ACE level and peripheral systolic
pressure [155, 239]. Although some data indicate a possible connection between
ACE and peripheral pressure, this may not hold true for central pressure. Our
study was the first to investigate the association between circulating ACE and
central hemodynamics. There was no association between circulating ACE and
RESULTS & DISCUSSION I 43
central aortic blood pressure, aortic augmentation or pulse pressure
amplification.
As a result of an increased afterload, chronically elevated blood pressure may
result in LVH. However, LVH has been observed in normotensive subjects, and
the degree of hypertension cannot explain the variance in LV mass [240, 241].
Although lowering of blood pressure has beneficial effects on LVH, clinical trials
have indicated that ACEi decrease LV mass more effectively than other
hypertensive drugs [242]. This finding has rendered the hypothesis that the
hemodynamic burden cannot be the sole determinant of LV structure and
function [243], and non-hemodynamic factors may also be of importance [19,
145, 146]. However, the possibility that this effect is due to a more pronounced
effect of ACEi on central pressure compared to peripheral pressure cannot be
excluded [237]. Our data showing an association between circulating ACE and LV
dysfunction, while there was no correlation between ACE and central blood
pressure supports the hypothesis of a pressure-independent role of ACE in
cardiac remodeling.
The ACE I/D polymorphism is probably one of the most frequently studied
candidate genes in CVD. Our study showed no difference in D allele frequency
between subjects with normal LV function and those with LV dysfunction.
However, a larger population with higher prevalence of LV dysfunction might
have been necessary to detect such an effect. The ACE D/D genotype has been
associated with increased risk of a number of different cardiac complications,
including myocardial infarction [58, 59], LVH [244] and heart failure [67],
although data are inconsistent [61, 183]. Today, there is no consensus regarding
the importance of the ACE genotype for CVD. The D/D genotype alone might not
be a risk factor for CVD, but together with other factors it may increase the risk
for cardiovascular complications and may be of particular importance in specific
subgroups.
The impact of the ACE I/D polymorphism on peripheral blood pressure has been
extensively studied, but the results are contradictory [61, 65, 66, 245]. However,
to our knowledge, only one previous study has investigated the impact of the ACE
I/D polymorphism on central pressure [218]. In agreement with Dima et al. [218],
we found no difference in central aortic blood pressure, aortic augmentation or
pulse pressure amplification between genotypes. Thus, at present, there is no
evidence for direct impact of the ACE I/D polymorphism or circulating ACE on
44 I RESULTS & DISCUSSION
central blood pressure. However, the relation between tissue ACE and central
hemodynamics is still unknown.
RESULTS & DISCUSSION I 45
LIMITATIONS
There are some limitations in our studies that should be pointed out. A weakness
in paper II-IV is the relatively small sample size (n= 406-672), leading to a low
number of subjects in subgroups (with respect to e.g. gender, genotype, LV
function). Also, as in all population based studies in elderly, there might be a
survival bias in our study, as some subjects obviously died before initiation of the
study. A selection against a specific ACE genotype is however contradicted by the
fact that the population was in accordance with the Hardy Weinberg equilibrium.
We reported a positive association between number of cardiovascular risk factors
and circulating ACE (paper II), as well as an association between ACE and LV
dysfunction (paper IV). An up-regulation of ACE might be involved in the
pathogenesis of CVD. However, we cannot exclude the possibility that an
increased ACE level is a non-pathological phenomenon, resulting from e.g.
increased shedding of ACE from ACE containing cells.
Furthermore, the abdominal aortic wall properties were calculated using the
brachial blood pressure for calibration. This may introduce a systematic bias as
the diastolic pressure is slightly higher in the brachial artery compared to the
abdominal aorta. However, as no age- or gender-related differences have been
observed, this systematic bias should not affect comparative studies between
groups.
The SphygmoCor system uses a generalized transfer function in order to
determine central hemodynamics. Although this transfer function has been
validated in a number of different populations, it is not individualized and may
thus not be accurate for all subjects. Furthermore, a second limitation is the
calibration of the radial artery pulse wave with brachial pressure. This may
introduce an error as it neglect the brachial-to-radial pressure amplification.
However, as the pressure differences in the arterial tree decrease with age, this
bias is probably less pronounced in elderly subjects.
46 I LIMITATIONS
CONCLUSIONS
Based on results from paper I-IV, the following conclusions can be drawn:
Nicotine and nicotine metabolites induce an increased ACE activity in cultured
human endothelial cells (HUVECs), and act primarily by affecting the enzymatic
activity, but may also induce an increased expression. The regulation of ACE by
nicotine seems to be restricted to ACE located in cells/tissues. There is an
individual variation in nicotine/nicotine metabolite induced up-regulation of
ACE, that appears to be independent of the ACE I/D polymorphism.
In elderly men, cardiovascular risk factors (such as smoking and diabetes) are
associated with higher levels of circulating ACE in men. The previously reported
association between the D/D genotype and CVD was not confirmed. Increased
ACE level may represent one of the cellular mechanisms involved in producing
the vascular damage associated with cardiovascular risk factors.
Men carrying the ACE D allele have higher abdominal aortic stiffness compared to
men carrying the I/I genotype. However, there is no association between
circulating ACE level and aortic stiffness, suggesting that the effect of the D allele
is due to factors other than elevated levels of circulating ACE. Increased
abdominal aortic stiffness suggests impaired vessel wall integrity, which
combined with local hemodynamic and/or inflammatory factors may have a role
in aneurysm formation.
Subjects with LV dysfunction have higher levels of circulating ACE compared to
those with normal LV function, and there is a relationship between circulating
ACE and degree of LV dysfunction. ACE might play a role in the pathogenesis of
left ventricular dysfunction and our findings suggest a direct effect on the heart
rather than affecting central blood pressure.
CONCLUSIONS I 47
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to everyone who contributed and
supported me throughout this work.
Especially, I would like to thank…
Toste Länne, my supervisor, for making it possible for me to continue with my
PhD-studies, and for all the support and encouragement along the way.
Karin Persson, my co-supervisor, for introducing me to an exciting scientific
field and for sharing your great knowledge in cardiovascular pharmacology.
Urban Alehagen, my co-supervisor, for support, good advices, and for “pushing”
me when needed. Thank you for giving me an insight into the clinical area.
My co-authors and co-workers…
Hanna Björck, with whom I’ve shared both ups and downs. Thank you for
valuable input on papers as well as thesis, for being a good friend and for all the
enjoyable “fungus experiences” (in Sweden as well as in Italy).
Rachel DeBasso and Ulf Dahlström, for stimulating collaboration, good
scientific advices and constructive criticism when preparing the manuscripts.
Christina Svensson and Elisabeth Kindberg, for performing the ultrasound
examinations and the blood pressure measurements in paper III and IV.
My colleagues and friends, especially…
Caroline Skoglund, for being a good friend, someone you can always count on.
Thank you for nice dinner parties, pleasant shoe-shopping, and for the crazy flea
market experience in Norrköping.
ACKNOWLEDGEMENTS I 49
Andreas Eriksson, who is always up for a challenge, I’ll never get tired of beating
you! Also, thank you for putting up with all the practical jokes and for sharing
your initials.
Ann-Charlotte Svensson Holm, my cell culture- and work out partner (for a
while at least). Thank you for all the encouragements!
Henrik Gréen, for always helping out with anything… (especially if it concerns
pharmacogenetics).
Anita Thunberg, for helping out with all practical issues.
Ida Bergström, Simon Jönsson and all the other friendly people at
“Farmakologen” who makes working much more enjoyable.
Stefan Klintström, and all the PhD students in Fourm Scientium.
My friends from the time as a Medical Biology student, Stina, Micke, Fredrik,
Sofia, Petter and Matilda for all the good times we spent together.
Till sist vill jag också rikta ett stort tack till min familj och mina vänner som
förgyller livet utanför forskarvärlden…
Mina underbara barndomsvänner Sandra, Helena, Jejja, Cilla och Sofie, det är
alltid lika kul att träffa er, tack för att ni finns där!
Hela ”Malmö-familjen” för att ni intresserar er för det jag gör och för att ni
välkomnat mig in i er familj.
Mamma, Pappa och Erika, för att ni tror på mig och stöttar mig i alla lägen. Ni
har gett mig de bästa förutsättningarna i livet och jag hoppas att jag gör er stolta.
Andy, min skatt… för allt stöd och uppmuntran och för att du alltid finns där för
mig. Tu sei tutto per me!
50 I ACKNOWLEDGEMENTS
This work was supported by grants from:
the Swedish Research Council, the Swedish Heart-Lung Foundation,
Cardiovascular Inflammatory Research Centre (CIRC), the Medical Research
Council of South East Sweden, the Medical Advisory Council, County Council of
Östergötland, Elanora Demoroutis Foundation, the Heart Foundation, the Health
Foundation, Sigrud and Elsa Golje Memorial Foundation and Lars Hierta
Memorial Foundation.
ACKNOWLEDGEMENTS I 51
REFERENCES
1.
Illustration: liver, kidney/adrenal gland and blood vessel adapted from "the
renin-angiotensin-aldosterone system" created by Rad A 2006
http://en.wikipedia.org/wiki/Renin-angiotensin_system
2. Urata H, Kinoshita A, Misono KS, Bumpus FM, Husain A. Identification of a
highly specific chymase as the major angiotensin II-forming enzyme in the
human heart. J Biol Chem. 1990;265(36):22348-57.
3. Hackenthal E, Hackenthal R, Hilgenfeldt U. Isorenin, pseudorenin, cathepsin
D and renin. A comparative enzymatic study of angiotensin-forming
enzymes. Biochim Biophys Acta. 1978;522(2):574-88.
4. Grise C, Boucher R, Thibault G, Genest J. Formation of angiotensin II by tonin
from partially purified human angiotensinogen. Can J Biochem.
1981;59(4):250-5.
5. Bader M. Tissue renin-angiotensin-aldosterone systems: Targets for
pharmacological therapy. Annu Rev Pharmacol Toxicol. 2010;50:439-65.
6. de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of
pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev.
2000;52(3):415-72.
7. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L,
Horiuchi M, et al. The angiotensin II type 2 (AT2) receptor antagonizes the
growth effects of the AT1 receptor: gain-of-function study using gene
transfer. Proc Natl Acad Sci U S A. 1995;92(23):10663-7.
8. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, et
al. Effects on blood pressure and exploratory behaviour of mice lacking
angiotensin II type-2 receptor. Nature. 1995;377(6551):748-50.
9. DiBona GF. Central sympathoexcitatory actions of angiotensin II: role of type
1 angiotensin II receptors. J Am Soc Nephrol. 1999;10 Suppl 11:S90-4.
10. Aguilera G, Kiss A. Regulation of the hypothalmic-pituitary-adrenal axis and
vasopressin secretion. Role of angiotensin II. Adv Exp Med Biol.
1996;396:105-12.
11. Fitzsimons JT. Angiotensin, thirst, and sodium appetite. Physiol Rev.
1998;78(3):583-686.
12. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not
hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res.
1988;62(4):749-56.
REFERENCES I 53
13. Campbell-Boswell M, Robertson AL, Jr. Effects of angiotensin II and
vasopressin on human smooth muscle cells in vitro. Exp Mol Pathol.
1981;35(2):265-76.
14. Naftilan AJ, Pratt RE, Dzau VJ. Induction of platelet-derived growth factor Achain and c-myc gene expressions by angiotensin II in cultured rat vascular
smooth muscle cells. J Clin Invest. 1989;83(4):1419-24.
15. Kato H, Suzuki H, Tajima S, Ogata Y, Tominaga T, Sato A, Saruta T.
Angiotensin II stimulates collagen synthesis in cultured vascular smooth
muscle cells. J Hypertens. 1991;9(1):17-22.
16. Tokimitsu I, Kato H, Wachi H, Tajima S. Elastin synthesis is inhibited by
angiotensin II but not by platelet-derived growth factor in arterial smooth
muscle cells. Biochim Biophys Acta. 1994;1207(1):68-73.
17. Koch-Weser J. Myocardial Actions of Angiotensin. Circ Res. 1964;14:337-44.
18. Dempsey PJ, McCallum ZT, Kent KM, Cooper T. Direct myocardial effects of
angiotensin II. Am J Physiol. 1971;220(2):477-81.
19. Baker KM, Aceto JF. Angiotensin II stimulation of protein synthesis and cell
growth in chick heart cells. Am J Physiol. 1990;259(2 Pt 2):H610-8.
20. Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, Baker KM.
Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res.
1993;72(6):1245-54.
21. Grafe M, Auch-Schwelk W, Zakrzewicz A, Regitz-Zagrosek V, Bartsch P, Graf
K, Loebe M, et al. Angiotensin II-induced leukocyte adhesion on human
coronary endothelial cells is mediated by E-selectin. Circ Res.
1997;81(5):804-11.
22. Pastore L, Tessitore A, Martinotti S, Toniato E, Alesse E, Bravi MC, Ferri C, et
al. Angiotensin II stimulates intercellular adhesion molecule-1 (ICAM-1)
expression by human vascular endothelial cells and increases soluble ICAM1 release in vivo. Circulation. 1999;100(15):1646-52.
23. Pueyo ME, Gonzalez W, Nicoletti A, Savoie F, Arnal JF, Michel JB. Angiotensin
II stimulates endothelial vascular cell adhesion molecule-1 via nuclear
factor-kappaB activation induced by intracellular oxidative stress.
Arterioscler Thromb Vasc Biol. 2000;20(3):645-51.
24. Fernandez-Castelo S, Arzt ES, Pesce A, Criscuolo ME, Diaz A, Finkielman S,
Nahmod VE. Angiotensin II regulates interferon-gamma production. J
Interferon Res. 1987;7(3):261-8.
25. Guo F, Chen XL, Wang F, Liang X, Sun YX, Wang YJ. Role of Angiotensin II
Type 1 Receptor in Angiotensin II-Induced Cytokine Production in
54 I REFERENCES
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Macrophages. J Interferon Cytokine Res. 2011;Epub ahead of print,
doi:10.1089/jir.2010.0073
Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II
stimulates NADH and NADPH oxidase activity in cultured vascular smooth
muscle cells. Circ Res. 1994;74(6):1141-8.
Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, Mugge A, Daniel
WG. Angiotensin II-induced superoxide anion generation in human vascular
endothelial cells: role of membrane-bound NADH-/NADPH-oxidases.
Cardiovasc Res. 1999;44(1):215-22.
Wolf RL, Mendlowitz M, Gitlow SE, Naftchi N. The metabolism of
angiotension II. Circ Res. 1962;11:195-208.
Haulica I, Bild W, Serban DN. Angiotensin peptides and their pleiotropic
actions. J Renin Angiotensin Aldosterone Syst. 2005;6(3):121-31.
Freeman EJ, Chisolm GM, Ferrario CM, Tallant EA. Angiotensin-(1-7) inhibits
vascular smooth muscle cell growth. Hypertension. 1996;28(1):104-8.
Benter IF, Ferrario CM, Morris M, Diz DI. Antihypertensive actions of
angiotensin-(1-7) in spontaneously hypertensive rats. Am J Physiol.
1995;269(1 Pt 2):H313-9.
Jaiswal N, Diz DI, Chappell MC, Khosla MC, Ferrario CM. Stimulation of
endothelial cell prostaglandin production by angiotensin peptides.
Characterization of receptors. Hypertension. 1992;19(2 Suppl):II49-55.
Ferrario CM, Chappell MC, Tallant EA, Brosnihan KB, Diz DI.
Counterregulatory actions of angiotensin-(1-7). Hypertension. 1997;30(3 Pt
2):535-41.
Rice GI, Thomas DA, Grant PJ, Turner AJ, Hooper NM. Evaluation of
angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in
angiotensin peptide metabolism. Biochem J. 2004;383(Pt 1):45-51.
Chappell MC, Pirro NT, Sykes A, Ferrario CM. Metabolism of angiotensin-(17) by angiotensin-converting enzyme. Hypertension. 1998;31(1 Pt 2):362-7.
Lieberman J, Sastre A. Angiotensin-converting enzyme activity in
postmortem human tissues. Lab Invest. 1983;48(6):711-7.
Ehlers MR, Fox EA, Strydom DJ, Riordan JF. Molecular cloning of human
testicular angiotensin-converting enzyme: the testis isozyme is identical to
the C-terminal half of endothelial angiotensin-converting enzyme. Proc Natl
Acad Sci U S A. 1989;86(20):7741-5.
Ryan JW, Ryan US, Schultz DR, Whitaker C, Chung A. Subcellular localization
of pulmonary antiotensin-converting enzyme (kininase II). Biochem J.
1975;146(2):497-9.
REFERENCES I 55
39. Costerousse O, Allegrini J, Lopez M, Alhenc-Gelas F. Angiotensin I-converting
enzyme in human circulating mononuclear cells: genetic polymorphism of
expression in T-lymphocytes. Biochem J. 1993;290 ( Pt 1):33-40.
40. Friedland J, Setton C, Silverstein E. Induction of angiotensin converting
enzyme in human monocytes in culture. Biochem Biophys Res Commun.
1978;83(3):843-9.
41. Danser AH, Schalekamp MA, Bax WA, van den Brink AM, Saxena PR, Riegger
GA, Schunkert H. Angiotensin-converting enzyme in the human heart. Effect
of the deletion/insertion polymorphism. Circulation. 1995;92(6):1387-8.
42. Diet F, Pratt RE, Berry GJ, Momose N, Gibbons GH, Dzau VJ. Increased
accumulation of tissue ACE in human atherosclerotic coronary artery
disease. Circulation. 1996;94(11):2756-67.
43. Wei L, Alhenc-Gelas F, Corvol P, Clauser E. The two homologous domains of
human angiotensin I-converting enzyme are both catalytically active. J Biol
Chem. 1991;266(14):9002-8.
44. Soubrier F, Alhenc-Gelas F, Hubert C, Allegrini J, John M, Tregear G, Corvol P.
Two putative active centers in human angiotensin I-converting enzyme
revealed by molecular cloning. In: Proc Natl Acad Sci U S A; 1988. p. 9386-90
45. Oppong SY, Hooper NM. Characterization of a secretase activity which
releases angiotensin-converting enzyme from the membrane. Biochem J.
1993;292 (Pt 2):597-603.
46. Beldent V, Michaud A, Wei L, Chauvet MT, Corvol P. Proteolytic release of
human angiotensin-converting enzyme. Localization of the cleavage site. J
Biol Chem. 1993;268(35):26428-34.
47. Yang HY, Erdos EG, Levin Y. A dipeptidyl carboxypeptidase that converts
angiotensin I and inactivates bradykinin. Biochim Biophys Acta.
1970;214(2):374-6.
48. Deddish PA, Jackman HL, Skidgel RA, Erdos EG. Differences in the hydrolysis
of enkephalin congeners by the two domains of angiotensin converting
enzyme. Biochem Pharmacol. 1997;53(10):1459-63.
49. Skidgel RA, Engelbrecht S, Johnson AR, Erdos EG. Hydrolysis of substance p
and neurotensin by converting enzyme and neutral endopeptidase. Peptides.
1984;5(4):769-76.
50. Hubert C, Houot AM, Corvol P, Soubrier F. Structure of the angiotensin Iconverting enzyme gene. Two alternate promoters correspond to
evolutionary steps of a duplicated gene. J Biol Chem. 1991;266(23):1537783.
56 I REFERENCES
51. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An
insertion/deletion polymorphism in the angiotensin I-converting enzyme
gene accounting for half the variance of serum enzyme levels. J Clin Invest.
1990;86(4):1343-6.
52. Cambien F, Costerousse O, Tiret L, Poirier O, Lecerf L, Gonzales MF, Evans A,
et al. Plasma level and gene polymorphism of angiotensin-converting
enzyme in relation to myocardial infarction. Circulation. 1994;90(2):669-76.
53. Villard E, Tiret L, Visvikis S, Rakotovao R, Cambien F, Soubrier F.
Identification of new polymorphisms of the angiotensin I-converting enzyme
(ACE) gene, and study of their relationship to plasma ACE levels by two-QTL
segregation-linkage analysis. Am J Hum Genet. 1996;58(6):1268-78.
54. Rieder MJ, Taylor SL, Clark AG, Nickerson DA. Sequence variation in the
human angiotensin converting enzyme. Nat Genet. 1999;22(1):59-62.
55. Ueda S, Elliott HL, Morton JJ, Connell JM. Enhanced pressor response to
angiotensin I in normotensive men with the deletion genotype (DD) for
angiotensin-converting enzyme. Hypertension. 1995;25(6):1266-9.
56. Danser AH, Deinum J, Osterop AP, Admiraal PJ, Schalekamp MA. Angiotensin
I to angiotensin II conversion in the human forearm and leg. Effect of the
angiotensin converting enzyme gene insertion/deletion polymorphism. J
Hypertens. 1999;17(12 Pt 2):1867-72.
57. van Dijk MA, Kroon I, Kamper AM, Boomsma F, Danser AH, Chang PC. The
angiotensin-converting enzyme gene polymorphism and responses to
angiotensins and bradykinin in the human forearm. J Cardiovasc Pharmacol.
2000;35(3):484-90.
58. Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D, Luc G, et al.
Deletion polymorphism in the gene for angiotensin-converting enzyme is a
potent risk factor for myocardial infarction. Nature. 1992;359(6396):641-4.
59. Wang XL, McCredie RM, Wilcken DE. Genotype distribution of angiotensinconverting enzyme polymorphism in Australian healthy and coronary
populations and relevance to myocardial infarction and coronary artery
disease. Arterioscler Thromb Vasc Biol. 1996;16(1):115-9.
60. Leatham E, Barley J, Redwood S, Hussein W, Carter N, Jeffery S, Bath PM, et
al. Angiotensin-1 converting enzyme (ACE) polymorphism in patients
presenting with myocardial infarction or unstable angina. J Hum Hypertens.
1994;8(8):635-8.
61. Agerholm-Larsen B, Nordestgaard BG, Steffensen R, Sorensen TI, Jensen G,
Tybjaerg-Hansen A. ACE gene polymorphism: ischemic heart disease and
longevity in 10,150 individuals. A case-referent and retrospective cohort
REFERENCES I 57
62.
63.
64.
65.
66.
67.
68.
69.
70.
study based on the Copenhagen City Heart Study. Circulation.
1997;95(10):2358-67.
Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F,
Buring J, et al. A prospective evaluation of an angiotensin-convertingenzyme gene polymorphism and the risk of ischemic heart disease. N Engl J
Med. 1995;332(11):706-11.
Mattu RK, Needham EW, Galton DJ, Frangos E, Clark AJ, Caulfield M. A DNA
variant at the angiotensin-converting enzyme gene locus associates with
coronary artery disease in the Caerphilly Heart Study. Circulation.
1995;91(2):270-4.
Gardemann A, Weiss T, Schwartz O, Eberbach A, Katz N, Hehrlein FW,
Tillmanns H, et al. Gene polymorphism but not catalytic activity of
angiotensin I-converting enzyme is associated with coronary artery disease
and myocardial infarction in low-risk patients. Circulation.
1995;92(10):2796-9.
Kario K, Hoshide S, Umeda Y, Sato Y, Ikeda U, Nishiuma S, Matsuo M, et al.
Angiotensinogen and angiotensin-converting enzyme genotypes, and day
and night blood pressures in elderly Japanese hypertensives. Hypertens Res.
1999;22(2):95-103.
O'Donnell CJ, Lindpaintner K, Larson MG, Rao VS, Ordovas JM, Schaefer EJ,
Myers RH, et al. Evidence for association and genetic linkage of the
angiotensin-converting enzyme locus with hypertension and blood pressure
in men but not women in the Framingham Heart Study. Circulation.
1998;97(18):1766-72.
Schut AF, Bleumink GS, Stricker BH, Hofman A, Witteman JC, Pols HA,
Deckers JW, et al. Angiotensin converting enzyme insertion/deletion
polymorphism and the risk of heart failure in hypertensive subjects. Eur
Heart J. 2004;25(23):2143-8.
Dux S, Aron N, Boner G, Carmel A, Yaron A, Rosenfeld JB. Serum angiotensin
converting enzyme activity in normal adults and patients with different
types of hypertension. Isr J Med Sci. 1984;20(12):1138-42.
Saijonmaa O, Nyman T, Fyhrquist F. Regulation of angiotensin-converting
enzyme production by nicotine in human endothelial cells. Am J Physiol
Heart Circ Physiol. 2005;289(5):H2000-4.
Gallagher PE, Li P, Lenhart JR, Chappell MC, Brosnihan KB. Estrogen
regulation of angiotensin-converting enzyme mRNA. Hypertension.
1999;33(1 Pt 2):323-8.
58 I REFERENCES
71. Dasarathy Y, Lanzillo JJ, Fanburg BL. Stimulation of bovine pulmonary artery
endothelial cell ACE by dexamethasone: involvement of steroid receptors.
Am J Physiol. 1992;263(6 Pt 1):L645-9.
72. Krulewitz AH, Baur WE, Fanburg BL. Hormonal influence on endothelial cell
angiotensin-converting enzyme activity. Am J Physiol. 1984;247(3 Pt
1):C163-8.
73. Hilgenfeldt U, Puschner T, Riester U, Finsterle J, Hilgenfeldt J, Ritz E. Low-salt
diet downregulates plasma but not tissue kallikrein-kinin system. Am J
Physiol. 1998;275(1 Pt 2):F88-93.
74. Kokubu T, Ueda E, Ono M, Kawabe T, Hayashi Y, Kan T. Effects of captopril
(SQ 14, 225) on the renin-angiotensin-aldosterone system in normal rats.
Eur J Pharmacol. 1980;62(4):269-75.
75. Mizuno K, Gotoh M, Matsui J, Nagasawa S, Fukuchi S. Acute effects of
captopril on serum angiotensin-converting enzyme activity, the reninaldosterone system and blood pressure in patients with sarcoidosis. Tohoku
J Exp Med. 1983;140(1):107-8.
76. Fyhrquist F, Hortling L, Gronhagen-Riska C. Induction of angiotensin Iconverting enzyme by captopril in cultured human endothelial cells. J Clin
Endocrinol Metab. 1982;55(4):783-6.
77. Fyhrquist F, Gronhagen-Riska C, Hortling L, Forslund T, Tikkanen I, Klockars
M. The induction of angiotensin converting enzyme by its inhibitors. Clin Exp
Hypertens A. 1983;5(7-8):1319-30.
78. Singh R, Kalra OP, Sarma PU, Prabhu KM. Effect of captopril on serum
angiotensin converting enzyme and blood pressure in hypertensive patients.
J Assoc Physicians India. 1996;44(2):109-11.
79. Ackermann A, Fernandez-Alfonso MS, Sanchez de Rojas R, Ortega T, Paul M,
Gonzalez C. Modulation of angiotensin-converting enzyme by nitric oxide. Br
J Pharmacol. 1998;124(2):291-8.
80. Persson K, Säfholm AC, Andersson RG, Ahlner J. Glyceryl trinitrate-induced
angiotensin-converting enzyme (ACE) inhibition in healthy volunteers is
dependent on ACE genotype. Can J Physiol Pharmacol. 2005;83(12):1117-22.
81. Gaertner R, Prunier F, Philippe M, Louedec L, Mercadier JJ, Michel JB. Scar
and pulmonary expression and shedding of ACE in rat myocardial infarction.
Am J Physiol Heart Circ Physiol. 2002;283(1):H156-64.
82. Yildiz D. Nicotine, its metabolism and an overview of its biological effects.
Toxicon. 2004;43(6):619-32.
83. Nakajima M, Kwon JT, Tanaka N, Zenta T, Yamamoto Y, Yamamoto H,
Yamazaki H, et al. Relationship between interindividual differences in
REFERENCES I 59
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
nicotine metabolism and CYP2A6 genetic polymorphism in humans. Clin
Pharmacol Ther. 2001;69(1):72-8.
Benowitz NL, Jacob P, 3rd, Fong I, Gupta S. Nicotine metabolic profile in man:
comparison of cigarette smoking and transdermal nicotine. J PharmaPrev
Medcol Exp Ther. 1994;268(1):296-303.
Benowitz NL, Jacob P, 3rd. Metabolism of nicotine to cotinine studied by a
dual stable isotope method. Clin Pharmacol Ther. 1994;56(5):483-93.
Benowitz NL, Kuyt F, Jacob P, 3rd. Circadian blood nicotine concentrations
during cigarette smoking. Clin Pharmacol Ther. 1982;32(6):758-64.
Benowitz NL, Jacob P, 3rd. Trans-3'-hydroxycotinine: disposition kinetics,
effects and plasma levels during cigarette smoking. Br J Clin Pharmacol.
2001;51(1):53-9.
Ambrose JA, Barua RS. The pathophysiology of cigarette smoking and
cardiovascular disease: an update. J Am Coll Cardiol. 2004;43(10):1731-7.
Benowitz NL, Gourlay SG. Cardiovascular toxicity of nicotine: implications
for nicotine replacement therapy. J Am Coll Cardiol. 1997;29(7):1422-31.
Benowitz NL. The role of nicotine in smoking-related cardiovascular disease.
Prev Med. 1997;26(4):412-7.
Benowitz NL. Pharmacology of nicotine: addiction and therapeutics. Annu
Rev Pharmacol Toxicol. 1996;36:597-613.
Cluette-Brown J, Mulligan J, Doyle K, Hagan S, Osmolski T, Hojnacki J. Oral
nicotine induces an atherogenic lipoprotein profile. Proc Soc Exp Biol Med.
1986;182(3):409-13.
Stefanovich V, Gore I, Kajiyama G, Iwanaga Y. The effect of nicotine on
dietary atherogenesis in rabbits. Exp Mol Pathol. 1969;11(1):71-81.
Strohschneider T, Oberhoff M, Hanke H, Hannekum A, Karsch KR. Effect of
chronic nicotine delivery on the proliferation rate of endothelial and smooth
muscle cells in experimentally induced vascular wall plaques. Clin Investig.
1994;72(11):908-12.
Neunteufl T, Heher S, Kostner K, Mitulovic G, Lehr S, Khoschsorur G, Schmid
RW, et al. Contribution of nicotine to acute endothelial dysfunction in longterm smokers. J Am Coll Cardiol. 2002;39(2):251-6.
Mayhan WG, Patel KP. Effect of nicotine on endothelium-dependent
arteriolar dilatation in vivo. Am J Physiol. 1997;272(5 Pt 2):H2337-42.
Chalon S, Moreno H, Jr., Benowitz NL, Hoffman BB, Blaschke TF. Nicotine
impairs endothelium-dependent dilatation in human veins in vivo. Clin
Pharmacol Ther. 2000;67(4):391-7.
60 I REFERENCES
98. Cucina A, Sapienza P, Borrelli V, Corvino V, Foresi G, Randone B, Cavallaro A,
et al. Nicotine reorganizes cytoskeleton of vascular endothelial cell through
platelet-derived growth factor BB. J Surg Res. 2000;92(2):233-8.
99. Villablanca AC. Nicotine stimulates DNA synthesis and proliferation in
vascular endothelial cells in vitro. J Appl Physiol. 1998;84(6):2089-98.
100. Rohani M, Agewall S. Oral snuff impairs endothelial function in healthy snuff
users. J Intern Med. 2004;255(3):379-83.
101. Hergens MP, Alfredsson L, Bolinder G, Lambe M, Pershagen G, Ye W. Longterm use of Swedish moist snuff and the risk of myocardial infarction
amongst men. J Intern Med. 2007;262(3):351-9.
102. Huhtasaari F, Lundberg V, Eliasson M, Janlert U, Asplund K. Smokeless
tobacco as a possible risk factor for myocardial infarction: a populationbased study in middle-aged men. J Am Coll Cardiol. 1999;34(6):1784-90.
103. Norman PE, Powell JT. Site specificity of aneurysmal disease. Circulation.
2010;121(4):560-8.
104. Ruddy JM, Jones JA, Spinale FG, Ikonomidis JS. Regional heterogeneity within
the aorta: relevance to aneurysm disease. J Thorac Cardiovasc Surg.
2008;136(5):1123-30.
105. Wolinsky H, Glagov S. Comparison of abdominal and thoracic aortic medial
structure in mammals. Deviation of man from the usual pattern. Circ Res.
1969;25(6):677-86.
106. Veller MG, Fisher CM, Nicolaides AN, Renton S, Geroulakos G, Stafford NJ,
Sarker A, et al. Measurement of the ultrasonic intima-media complex
thickness in normal subjects. J Vasc Surg. 1993;17(4):719-25.
107. Virmani R, Avolio AP, Mergner WJ, Robinowitz M, Herderick EE, Cornhill JF,
Guo SY, et al. Effect of aging on aortic morphology in populations with high
and low prevalence of hypertension and atherosclerosis. Comparison
between occidental and Chinese communities. Am J Pathol.
1991;139(5):1119-29.
108. Länne T, Hansen F, Mangell P, Sonesson B. Differences in mechanical
properties of the common carotid artery and abdominal aorta in healthy
males. J Vasc Surg. 1994;20(2):218-25.
109. Latham RD, Westerhof N, Sipkema P, Rubal BJ, Reuderink P, Murgo JP.
Regional wave travel and reflections along the human aorta: a study with six
simultaneous micromanometric pressures. Circulation. 1985;72(6):1257-69.
110. Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC.
Suggested standards for reporting on arterial aneurysms. Subcommittee on
Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on
REFERENCES I 61
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
Reporting Standards, Society for Vascular Surgery and North American
Chapter, International Society for Cardiovascular Surgery. J Vasc Surg.
1991;13(3):452-8.
Collaborative Aneurysm Screening Study Group. A comparative study of the
prevalence of abdominal aortic aneurysms in the United Kingdom, Denmark,
and Australia. J Med Screen. 2001;8(1):46-50.
Thomas PR, Stewart RD. Abdominal aortic aneurysm. Br J Surg.
1988;75(8):733-6.
Länne T, Sonesson B, Bergqvist D, Bengtsson H, Gustafsson D. Diameter and
compliance in the male human abdominal aorta: influence of age and aortic
aneurysm. Eur J Vasc Surg. 1992;6(2):178-84.
MacSweeney ST, Young G, Greenhalgh RM, Powell JT. Mechanical properties
of the aneurysmal aorta. Br J Surg. 1992;79(12):1281-4.
Willum-Hansen T, Staessen JA, Torp-Pedersen C, Rasmussen S, Thijs L, Ibsen
H, Jeppesen J. Prognostic value of aortic pulse wave velocity as index of
arterial stiffness in the general population. Circulation. 2006;113(5):664-70.
Boutouyrie P, Tropeano AI, Asmar R, Gautier I, Benetos A, Lacolley P,
Laurent S. Aortic stiffness is an independent predictor of primary coronary
events in hypertensive patients: a longitudinal study. Hypertension.
2002;39(1):10-5.
Hoffman AS, Grande LA, Park JB. Sequential enzymolysis of human aorta and
resultant stress-strain behavior. Biomater Med Devices Artif Organs.
1977;5(2):121-45.
Roach MR, Burton AC. The reason for the shape of the distensibility curves of
arteries. Can J Biochem Physiol. 1957;35(8):681-90.
Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D,
Pannier B, et al. Expert consensus document on arterial stiffness:
methodological issues and clinical applications. Eur Heart J.
2006;27(21):2588-605.
Fischer GM, Llaurado JG. Collagen and elastin content in canine arteries
selected from functionally different vascular beds. Circ Res. 1966;19(2):3949.
Benetos A, Laurent S, Hoeks AP, Boutouyrie PH, Safar ME. Arterial
alterations with aging and high blood pressure. A noninvasive study of
carotid and femoral arteries. Arterioscler Thromb. 1993;13(1):90-7.
Mahmud A, Feely J. Effect of smoking on arterial stiffness and pulse pressure
amplification. Hypertension. 2003;41(1):183-7.
62 I REFERENCES
123. Åstrand H, Stålhand J, Karlsson J, Karlsson M, Sonesson B, Länne T. In vivo
estimation of the contribution of elastin and collagen to the mechanical
properties in the human abdominal aorta: effect of age and sex. J Appl
Physiol. 2011;110(1):176-87.
124. Reneman RS, van Merode T, Hick P, Muytjens AM, Hoeks AP. Age-related
changes in carotid artery wall properties in men. Ultrasound Med Biol.
1986;12(6):465-71.
125. Nichols W, O´Rourke M. Ageing. In: McDonald´s blood flow in arteries. 5th
ed. Hodder Arnold, London: 2005. p. 341-367
126. Hansen F, Mangell P, Sonesson B, Länne T. Diameter and compliance in the
human common carotid artery-variations with age and sex. Ultrasound Med
Biol. 1995;21(1):1-9.
127. Mitchell GF, Parise H, Benjamin EJ, Larson MG, Keyes MJ, Vita JA, Vasan RS, et
al. Changes in arterial stiffness and wave reflection with advancing age in
healthy men and women: the Framingham Heart Study. Hypertension.
2004;43(6):1239-45.
128. Sonesson B, Hansen F, Stale H, Länne T. Compliance and diameter in the
human abdominal aorta-the influence of age and sex. Eur J Vasc Surg.
1993;7(6):690-7.
129. van der Heijden-Spek JJ, Staessen JA, Fagard RH, Hoeks AP, Boudier HA, van
Bortel LM. Effect of age on brachial artery wall properties differs from the
aorta and is gender dependent: a population study. Hypertension.
2000;35(2):637-42.
130. Åstrand H, Ryden-Ahlgren A, Sandgren T, Länne T. Age-related increase in
wall stress of the human abdominal aorta: an in vivo study. J Vasc Surg.
2005;42(5):926-31.
131. Laurent S, Boutouyrie P, Lacolley P. Structural and genetic bases of arterial
stiffness. Hypertension. 2005;45(6):1050-5.
132. Powell JT, Turner RJ, Sian M, Debasso R, Länne T. Influence of fibrillin-1
genotype on the aortic stiffness in men. J Appl Physiol. 2005;99(3):1036-40.
133. Sonesson B, Hansen F, Länne T. Abnormal mechanical properties of the aorta
in Marfan's syndrome. Eur J Vasc Surg. 1994;8(5):595-601.
134. Medley TL, Cole TJ, Dart AM, Gatzka CD, Kingwell BA. Matrix
metalloproteinase-9 genotype influences large artery stiffness through
effects on aortic gene and protein expression. Arterioscler Thromb Vasc Biol.
2004;24(8):1479-84.
135. Medley TL, Kingwell BA, Gatzka CD, Pillay P, Cole TJ. Matrix
metalloproteinase-3 genotype contributes to age-related aortic stiffening
REFERENCES I 63
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
through modulation of gene and protein expression. Circ Res.
2003;92(11):1254-61.
Björck HM, Länne T, Alehagen U, Persson K, Rundkvist L, Hamsten A,
Dahlström U, et al. Association of genetic variation on chromosome 9p21.3
and arterial stiffness. J Intern Med. 2009;265(3):373-81.
Nichols W, O´Rourke M. Wave reflections. In: McDonald´s blood flow in
arteries. 5th ed. Hodder Arnold, London: 2005. p. 193-213
Segers P, Mahieu D, Kips J, Rietzschel E, De Buyzere M, De Bacquer D,
Bekaert S, et al. Amplification of the pressure pulse in the upper limb in
healthy, middle-aged men and women. Hypertension. 2009;54(2):414-20.
Roman MJ, Devereux RB, Kizer JR, Lee ET, Galloway JM, Ali T, Umans JG, et al.
Central pressure more strongly relates to vascular disease and outcome than
does brachial pressure: the Strong Heart Study. Hypertension.
2007;50(1):197-203.
Wang KL, Cheng HM, Chuang SY, Spurgeon HA, Ting CT, Lakatta EG, Yin FC,
et al. Central or peripheral systolic or pulse pressure: which best relates to
target organs and future mortality? J Hypertens. 2009;27(3):461-7.
McMurray JJ, Stewart S. Epidemiology, aetiology, and prognosis of heart
failure. Heart. 2000;83(5):596-602.
Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart.
2007;93(9):1137-46.
Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic
implications of echocardiographically determined left ventricular mass in
the Framingham Heart Study. N Engl J Med. 1990;322(22):1561-6.
Artham SM, Lavie CJ, Milani RV, Patel DA, Verma A, Ventura HO. Clinical
impact of left ventricular hypertrophy and implications for regression. Prog
Cardiovasc Dis. 2009;52(2):153-67.
Malmqvist K, Öhman KP, Lind L, Nyström F, Kahan T. Relationships between
left ventricular mass and the renin-angiotensin system, catecholamines,
insulin and leptin. J Intern Med. 2002;252(5):430-9.
Kelm M, Schafer S, Mingers S, Heydthausen M, Vogt M, Motz W, Strauer BE.
Left ventricular mass is linked to cardiac noradrenaline in normotensive and
hypertensive patients. J Hypertens. 1996;14(11):1357-64.
The SOLVD investigators. Effect of enalapril on survival in patients with
reduced left ventricular ejection fractions and congestive heart failure.
N Engl J Med. 1991;325(5):293-302.
The CONSENSUS Trial Study Group. Effects of enalapril on mortality in
severe congestive heart failure. Results of the Cooperative North
64 I REFERENCES
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
Scandinavian Enalapril Survival Study (CONSENSUS). N Engl J Med.
1987;316(23):1429-35.
Pitt B, Poole-Wilson PA, Segal R, Martinez FA, Dickstein K, Camm AJ,
Konstam MA, et al. Effect of losartan compared with captopril on mortality
in patients with symptomatic heart failure: randomised trial - the Losartan
Heart Failure Survival Study ELITE II. Lancet. 2000;355(9215):1582-7.
Pfeffer MA, Swedberg K, Granger CB, Held P, McMurray JJ, Michelson EL,
Olofsson B, et al. Effects of candesartan on mortality and morbidity in
patients with chronic heart failure: the CHARM-Overall programme. Lancet.
2003;362(9386):759-66.
Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial
cells derived from umbilical veins. Identification by morphologic and
immunologic criteria. J Clin Invest. 1973;52(11):2745-56.
Relou IA, Damen CA, van der Schaft DW, Groenewegen G, Griffioen AW.
Effect of culture conditions on endothelial cell growth and responsiveness.
Tissue Cell. 1998;30(5):525-30.
Balyasnikova IV, Danilov SM, Muzykantov VR, Fisher AB. Modulation of
angiotensin-converting enzyme in cultured human vascular endothelial
cells. In Vitro Cell Dev Biol Anim. 1998;34(7):545-54.
Alehagen U, Goetze JP, Dahlström U. Reference intervals and decision limits
for B-type natriuretic peptide (BNP) and its precursor (Nt-proBNP) in the
elderly. Clin Chim Acta. 2007;382(1-2):8-14.
Alhenc-Gelas F, Richard J, Courbon D, Warnet JM, Corvol P. Distribution of
plasma angiotensin I-converting enzyme levels in healthy men: relationship
to environmental and hormonal parameters. J Lab Clin Med. 1991;117(1):339.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the
insertion/deletion polymorphism of the human angiotensin converting
enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res.
1992;20(6):1433.
Shanmugam V, Sell KW, Saha BK. Mistyping ACE heterozygotes. PCR Methods
Appl. 1993;3(2):120-1.
Cheon KT, Choi KH, Lee HB, Park SK, Rhee YK, Lee YC. Gene polymorphisms
of endothelial nitric oxide synthase and angiotensin-converting enzyme in
patients with lung cancer. Lung. 2000;178(6):351-60.
Ueda S, Heeley RP, Lees KR, Elliott HL, Connell JM. Mistyping of the human
angiotensin-converting enzyme gene polymorphism: frequency, causes and
REFERENCES I 65
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
possible methods to avoid errors in typing. J Mol Endocrinol. 1996;17(1):2730.
Odawara M, Matsunuma A, Yamashita K. Mistyping frequency of the
angiotensin-converting enzyme gene polymorphism and an improved
method for its avoidance. Hum Genet. 1997;100(2):163-6.
Hoeks AP, Willekes C, Boutouyrie P, Brands PJ, Willigers JM, Reneman RS.
Automated detection of local artery wall thickness based on M-line signal
processing. Ultrasound Med Biol. 1997;23(7):1017-23.
Länne T, Stale H, Bengtsson H, Gustafsson D, Bergqvist D, Sonesson B,
Lecerof H, et al. Noninvasive measurement of diameter changes in the distal
abdominal aorta in man. Ultrasound Med Biol. 1992;18(5):451-7.
Sonesson B, Länne T, Vernersson E, Hansen F. Sex difference in the
mechanical properties of the abdominal aorta in human beings. J Vasc Surg.
1994;20(6):959-69.
Kawasaki T, Sasayama S, Yagi S, Asakawa T, Hirai T. Non-invasive
assessment of the age related changes in stiffness of major branches of the
human arteries. Cardiovasc Res. 1987;21(9):678-87.
Pauca AL, O'Rourke MF, Kon ND. Prospective evaluation of a method for
estimating ascending aortic pressure from the radial artery pressure
waveform. Hypertension. 2001;38(4):932-7.
Sharman JE, Lim R, Qasem AM, Coombes JS, Burgess MI, Franco J, Garrahy P,
et al. Validation of a generalized transfer function to noninvasively derive
central blood pressure during exercise. Hypertension. 2006;47(6):1203-8.
Hope SA, Tay DB, Meredith IT, Cameron JD. Use of arterial transfer functions
for the derivation of aortic waveform characteristics. J Hypertens.
2003;21(7):1299-305.
Davies JI, Band MM, Pringle S, Ogston S, Struthers AD. Peripheral blood
pressure measurement is as good as applanation tonometry at predicting
ascending aortic blood pressure. J Hypertens. 2003;21(3):571-6.
van Royen N, Jaffe CC, Krumholz HM, Johnson KM, Lynch PJ, Natale D,
Atkinson P, et al. Comparison and reproducibility of visual
echocardiographic and quantitative radionuclide left ventricular ejection
fractions. Am J Cardiol. 1996;77(10):843-50.
Jensen-Urstad K, Bouvier F, Hojer J, Ruiz H, Hulting J, Samad B, Thorstrand C,
et al. Comparison of different echocardiographic methods with radionuclide
imaging for measuring left ventricular ejection fraction during acute
myocardial infarction treated by thrombolytic therapy. Am J Cardiol.
1998;81(5):538-44.
66 I REFERENCES
171. Choy AM, Darbar D, Lang CC, Pringle TH, McNeill GP, Kennedy NS, Struthers
AD. Detection of left ventricular dysfunction after acute myocardial
infarction: comparison of clinical, echocardiographic, and neurohormonal
methods. Br Heart J. 1994;72(1):16-22.
172. Ekman I, Alehagen U, Dahlström U, Käll B, Nylander E. Semikvantitativ
vänsterkammarfunktionsklassificering istället för
ejektionsfractionsmätning, användbar för hjärtsviktsstudier hos äldre
patienter. Riksstämman. Dec 2000 Göteborg. Abstract
173. Zhang S, Day I, Ye S. Nicotine induced changes in gene expression by human
coronary artery endothelial cells. Atherosclerosis. 2001;154(2):277-83.
174. Sugiyama Y, Yotsumoto H, Takaku F. Increase of serum angiotensinconverting enzyme level after exposure to cigarette smoke and nicotine
infusion in dogs. Respiration. 1986;49(4):292-5.
175. Luo HL, Zang WJ, Lu J, Yu XJ, Lin YX, Cao YX. The protective effect of captopril
on nicotine-induced endothelial dysfunction in rat. Basic Clin Pharmacol
Toxicol. 2006;99(3):237-45.
176. Ueda S, Meredith PA, Morton JJ, Connell JM, Elliott HL. ACE (I/D) genotype as
a predictor of the magnitude and duration of the response to an ACE
inhibitor drug (enalaprilat) in humans. Circulation. 1998;98(20):2148-53.
177. Zhu X, Bouzekri N, Southam L, Cooper RS, Adeyemo A, McKenzie CA, Luke A,
et al. Linkage and association analysis of angiotensin I-converting enzyme
(ACE)-gene polymorphisms with ACE concentration and blood pressure. Am
J Hum Genet. 2001;68(5):1139-48.
178. Zhu X, McKenzie CA, Forrester T, Nickerson DA, Broeckel U, Schunkert H,
Doering A, et al. Localization of a small genomic region associated with
elevated ACE. Am J Hum Genet. 2000;67(5):1144-53.
179. McKenzie CA, Zhu X, Forrester TE, Luke A, Adeyemo AA, Bouzekri N, Cooper
RS. A genome-wide search replicates evidence of a quantitative trait locus
for circulating angiotensin I-converting enzyme (ACE) unlinked to the ACE
gene. BMC Med Genomics. 2008;1:23.
180. Saijonmaa O, Nyman T, Kosonen R, Fyhrquist F. Upregulation of angiotensinconverting enzyme by vascular endothelial growth factor. Am J Physiol Heart
Circ Physiol. 2001;280(2):H885-91.
181. Bloem LJ, Manatunga AK, Pratt JH. Racial difference in the relationship of an
angiotensin I-converting enzyme gene polymorphism to serum angiotensin
I-converting enzyme activity. Hypertension. 1996;27(1):62-6.
182. Lieberman J. Elevation of serum angiotensin-converting-enzyme (ACE) level
in sarcoidosis. Am J Med. 1975;59(3):365-72.
REFERENCES I 67
183. Jalil JE, Piddo AM, Cordova S, Chamorro G, Braun S, Jalil R, Vega J, et al.
Prevalence of the angiotensin I converting enzyme insertion/deletion
polymorphism, plasma angiotensin converting enzyme activity, and left
ventricular mass in a normotensive Chilean population. Am J Hypertens.
1999;12(7):697-704.
184. Sayed-Tabatabaei FA, Schut AF, Vasquez AA, Bertoli-Avella AM, Hofman A,
Witteman JC, van Duijn CM. Angiotensin converting enzyme gene
polymorphism and cardiovascular morbidity and mortality: the Rotterdam
Study. J Med Genet. 2005;42(1):26-30.
185. Sagnella GA, Rothwell MJ, Onipinla AK, Wicks PD, Cook DG, Cappuccio FP. A
population study of ethnic variations in the angiotensin-converting enzyme
I/D polymorphism: relationships with gender, hypertension and impaired
glucose metabolism. J Hypertens. 1999;17(5):657-64.
186. Barley J, Blackwood A, Miller M, Markandu ND, Carter ND, Jeffery S,
Cappuccio FP, et al. Angiotensin converting enzyme gene I/D polymorphism,
blood pressure and the renin-angiotensin system in Caucasian and AfroCaribbean peoples. J Hum Hypertens. 1996;10(1):31-5.
187. Mathew J, Basheeruddin K, Prabhakar S. Differences in frequency of the
deletion polymorphism of the angiotensin-converting enzyme gene in
different ethnic groups. Angiology. 2001;52(6):375-9.
188. Payne JR, Dhamrait SS, Gohlke P, Cooper J, Scott RA, Pitsiladis YP, Humphries
SE, et al. The impact of ACE genotype on serum ACE activity in a black South
African male population. Ann Hum Genet. 2007;71(Pt 1):1-7.
189. Rodriguez GE, Shin BC, Abernathy RS, Kendig EL, Jr. Serum angiotensinconverting enzyme activity in normal children and in those with sarcoidosis.
J Pediatr. 1981;99(1):68-72.
190. Erman A, Chen-Gal B, van Dijk DJ, Sulkes J, Kaplan B, Boner G, Neri A. Ovarian
angiotensin-converting enzyme activity in humans: relationship to estradiol,
age, and uterine pathology. J Clin Endocrinol Metab. 1996;81(3):1104-7.
191. Biller H, Zissel G, Ruprecht B, Nauck M, Busse Grawitz A, Muller-Quernheim
J. Genotype-corrected reference values for serum angiotensin-converting
enzyme. Eur Respir J. 2006;28(6):1085-90.
192. Noronha-Dutra AA, Epperlein MM, Woolf N. Effect of cigarette smoking on
cultured human endothelial cells. Cardiovasc Res. 1993;27(5):774-8.
193. Mizuno K, Yaginuma K, Hashimoto S, Toki T, Nakamura I, Fukuchi S. Acute
effect of cigarette smoking on serum angiotensin-converting enzyme activity
in normal man. Tohoku J Exp Med. 1982;137(1):113-4.
68 I REFERENCES
194. Ninomiya Y, Kioi S, Arakawa M. Serum angiotensin converting enzyme
activity in ex-smokers. Clin Chim Acta. 1987;164(2):223-6.
195. Haboubi NA, Bignell AH, Haboubi NY. Serum angiotensin converting enzyme
activity in cigarette smokers. Clin Chim Acta. 1986;154(1):69-72.
196. Sayed-Tabatabaei FA, Schut AF, Hofman A, Bertoli-Avella AM, Vergeer J,
Witteman JC, van Duijn CM. A study of gene--environment interaction on the
gene for angiotensin converting enzyme: a combined functional and
population based approach. J Med Genet. 2004;41(2):99-103.
197. Ljungberg LU, Persson K. Effect of nicotine and nicotine metabolites on
Angiotensin-converting enzyme in human endothelial cells. Endothelium.
2008;15(5):239-45.
198. Ustundag B, Canatan H, Cinkilinc N, Halifeoglu I, Bahcecioglu IH. Angiotensin
converting enzyme (ACE) activity levels in insulin-independent diabetes
mellitus and effect of ACE levels on diabetic patients with nephropathy. Cell
Biochem Funct. 2000;18(1):23-8.
199. Van Dyk DJ, Erman A, Erman T, Chen-Gal B, Sulkes J, Boner G. Increased
serum angiotensin converting enzyme activity in type I insulin-dependent
diabetes mellitus: its relation to metabolic control and diabetic
complications. Eur J Clin Invest. 1994;24(7):463-7.
200. Schernthaner G, Schwarzer C, Kuzmits R, Muller MM, Klemen U, Freyler H.
Increased angiotensin-converting enzyme activities in diabetes mellitus:
analysis of diabetes type, state of metabolic control and occurrence of
diabetic vascular disease. J Clin Pathol. 1984;37(3):307-12.
201. Wong TY, Chan JC, Szeto CC, Leung CB, Li PK. Clinical and biochemical
characteristics of type 2 diabetic patients on continuous ambulatory
peritoneal dialysis: relationships with insulin requirement. Am J Kidney Dis.
1999;34(3):514-20.
202. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R, Dagenais G. Effects of an
angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events
in high-risk patients. The Heart Outcomes Prevention Evaluation Study
Investigators. N Engl J Med. 2000;342(3):145-53.
203. Niskanen L, Hedner T, Hansson L, Lanke J, Niklason A. Reduced
cardiovascular morbidity and mortality in hypertensive diabetic patients on
first-line therapy with an ACE inhibitor compared with a diuretic/betablocker-based treatment regimen: a subanalysis of the Captopril Prevention
Project. Diabetes Care. 2001;24(12):2091-6.
204. Costerousse O, Allegrini J, Clozel JP, Menard J, Alhenc-Gelas F. Angiotensin Iconverting enzyme inhibition but not angiotensin II suppression alters
REFERENCES I 69
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
angiotensin I-converting enzyme gene expression in vessels and epithelia. J
Pharmacol Exp Ther. 1998;284(3):1180-7.
Fyhrquist F, Forslund T, Tikkanen I, Gronhagen-Riska C. Induction of
angiotensin I-converting enzyme rat lung with Captopril (SQ 14225). Eur J
Pharmacol. 1980;67(4):473-5.
Petrov VV, Fagard RH, Lijnen PJ. T-lymphocyte and plasma angiotensinconverting enzyme activity during enalapril and losartan administration in
humans. J Cardiovasc Pharmacol. 2001;38(4):578-83.
Kohlstedt K, Brandes RP, Muller-Esterl W, Busse R, Fleming I. Angiotensinconverting enzyme is involved in outside-in signaling in endothelial cells.
Circ Res. 2004;94(1):60-7.
Mahmud A, Feely J. Effect of angiotensin ii receptor blockade on arterial
stiffness: beyond blood pressure reduction. Am J Hypertens.
2002;15(12):1092-5.
London GM, Pannier B, Vicaut E, Guerin AP, Marchais SJ, Safar ME, Cuche JL.
Antihypertensive effects and arterial haemodynamic alterations during
angiotensin converting enzyme inhibition. J Hypertens. 1996;14(9):1139-46.
Nishimoto M, Takai S, Fukumoto H, Tsunemi K, Yuda A, Sawada Y, Yamada
M, et al. Increased local angiotensin II formation in aneurysmal aorta. Life
Sci. 2002;71(18):2195-205.
Daugherty A, Manning MW, Cassis LA. Angiotensin II promotes
atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J
Clin Invest. 2000;105(11):1605-12.
Balkestein EJ, Staessen JA, Wang JG, van Der Heijden-Spek JJ, Van Bortel LM,
Barlassina C, Bianchi G, et al. Carotid and femoral artery stiffness in relation
to three candidate genes in a white population. Hypertension.
2001;38(5):1190-7.
Mattace-Raso FU, van der Cammen TJ, Sayed-Tabatabaei FA, van Popele NM,
Asmar R, Schalekamp MA, Hofman A, et al. Angiotensin-converting enzyme
gene polymorphism and common carotid stiffness. The Rotterdam study.
Atherosclerosis. 2004;174(1):121-6.
Sie MP, Yazdanpanah M, Mattace-Raso FU, Uitterlinden AG, Hofman A, Hoeks
AP, Reneman RS, et al. Genetic variation in the renin-angiotensin system and
arterial stiffness. The Rotterdam Study. Clin Exp Hypertens. 2009;31(5):38999.
Taniwaki H, Kawagishi T, Emoto M, Shoji T, Hosoi M, Kogawa K, Nishizawa Y,
et al. Association of ACE gene polymorphism with arterial stiffness in
patients with type 2 diabetes. Diabetes Care. 1999;22(11):1858-64.
70 I REFERENCES
216. Lajemi M, Labat C, Gautier S, Lacolley P, Safar M, Asmar R, Cambien F, et al.
Angiotensin II type 1 receptor-153A/G and 1166A/C gene polymorphisms
and increase in aortic stiffness with age in hypertensive subjects. J
Hypertens. 2001;19(3):407-13.
217. Benetos A, Gautier S, Ricard S, Topouchian J, Asmar R, Poirier O, Larosa E, et
al. Influence of angiotensin-converting enzyme and angiotensin II type 1
receptor gene polymorphisms on aortic stiffness in normotensive and
hypertensive patients. Circulation. 1996;94(4):698-703.
218. Dima I, Vlachopoulos C, Alexopoulos N, Baou K, Vasiliadou C, Antoniades C,
Aznaouridis K, et al. Association of arterial stiffness with the angiotensinconverting enzyme gene polymorphism in healthy individuals. Am J
Hypertens. 2008;21(12):1354-8.
219. Jondeau G, Boutouyrie P, Lacolley P, Laloux B, Dubourg O, Bourdarias JP,
Laurent S. Central pulse pressure is a major determinant of ascending aorta
dilation in Marfan syndrome. Circulation. 1999;99(20):2677-81.
220. Mahmud A, Feely J. Reduction in arterial stiffness with angiotensin II
antagonist is comparable with and additive to ACE inhibition. Am J
Hypertens. 2002;15(4 Pt 1):321-5.
221. Fatini C, Pratesi G, Sofi F, Gensini F, Sticchi E, Lari B, Pulli R, et al. ACE DD
genotype: a predisposing factor for abdominal aortic aneurysm. Eur J Vasc
Endovasc Surg. 2005;29(3):227-32.
222. Pola R, Gaetani E, Santoliquido A, Gerardino L, Cattani P, Serricchio M, Tondi
P, et al. Abdominal aortic aneurysm in normotensive patients: association
with angiotensin-converting enzyme gene polymorphism. Eur J Vasc
Endovasc Surg. 2001;21(5):445-9.
223. Jones GT, Thompson AR, van Bockxmeer FM, Hafez H, Cooper JA, Golledge J,
Humphries SE, et al. Angiotensin II type 1 receptor 1166C polymorphism is
associated with abdominal aortic aneurysm in three independent cohorts.
Arterioscler Thromb Vasc Biol. 2008;28(4):764-70.
224. Korcz A, Mikolajczyk-Stecyna J, Gabriel M, Zowczak-Drabarczyk M,
Pawlaczyk K, Kalafirov M, Oszkinis G, et al. Angiotensin-converting enzyme
(ACE, I/D) gene polymorphism and susceptibility to abdominal aortic
aneurysm or aortoiliac occlusive disease. J Surg Res. 2009;153(1):76-82.
225. Abdulla J, Barlera S, Latini R, Kjoller-Hansen L, Sogaard P, Christensen E,
Kober L, et al. A systematic review: effect of angiotensin converting enzyme
inhibition on left ventricular volumes and ejection fraction in patients with a
myocardial infarction and in patients with left ventricular dysfunction. Eur J
Heart Fail. 2007;9(2):129-35.
REFERENCES I 71
226. Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS, Lorell BH. Increased
rat cardiac angiotensin converting enzyme activity and mRNA expression in
pressure overload left ventricular hypertrophy. Effects on coronary
resistance, contractility, and relaxation. J Clin Invest. 1990;86(6):1913-20.
227. Schunkert H, Jackson B, Tang SS, Schoen FJ, Smits JF, Apstein CS, Lorell BH.
Distribution and functional significance of cardiac angiotensin converting
enzyme in hypertrophied rat hearts. Circulation. 1993;87(4):1328-39.
228. Hirsch AT, Talsness CE, Schunkert H, Paul M, Dzau VJ. Tissue-specific
activation of cardiac angiotensin converting enzyme in experimental heart
failure. Circ Res. 1991;69(2):475-82.
229. Schunkert H, Hense HW, Muscholl M, Luchner A, Kurzinger S, Danser AH,
Riegger GA. Associations between circulating components of the reninangiotensin-aldosterone system and left ventricular mass. Heart.
1997;77(1):24-31.
230. Reneland R, Andren B, Lind L, Andersson PE, Hanni A, Lithell H. Circulating
angiotensin converting enzyme levels are increased in concentric, but not
eccentric, left ventricular hypertrophy in elderly men. J Hypertens.
1997;15(8):885-90.
231. Buck PC, Fernandes F, Arteaga E, Matsumoto AY, Araujo AQ, Oliveira EM,
Ianni BM, et al. Association of angiotensin-converting enzyme activity and
polymorphism with echocardiographic measures in familial and nonfamilial
hypertrophic cardiomyopathy. Braz J Med Biol Res. 2009;42(8):717-21.
232. Metzger R, Bohle RM, Chumachenko P, Danilov SM, Franke FE. CD143 in the
development of atherosclerosis. Atherosclerosis. 2000;150(1):21-31.
233. Hokimoto S, Yasue H, Fujimoto K, Yamamoto H, Nakao K, Kaikita K, Sakata R,
et al. Expression of angiotensin-converting enzyme in remaining viable
myocytes of human ventricles after myocardial infarction. Circulation.
1996;94(7):1513-8.
234. Pulla Reddy B, Srikanth Babu BM, Venkata Karunakar K, Yasovanthi J,
Munshi A, Sampath Kumar P, Sharath A, et al. Angiotensin-converting
enzyme gene variant and its levels: risk factors for myocardial infarction in a
South Indian population. Singapore Med J. 2010;51(7):576-81.
235. Davis GK, Millner RW, Roberts DH. Angiotensin converting enzyme (ACE)
gene expression in the human left ventricle: effect of ACE gene
insertion/deletion polymorphism and left ventricular function. Eur J Heart
Fail. 2000;2(3):253-6.
236. Studer R, Reinecke H, Muller B, Holtz J, Just H, Drexler H. Increased
angiotensin-I converting enzyme gene expression in the failing human heart.
72 I REFERENCES
237.
238.
239.
240.
241.
242.
243.
244.
245.
Quantification by competitive RNA polymerase chain reaction. J Clin Invest.
1994;94(1):301-10.
Morgan T, Lauri J, Bertram D, Anderson A. Effect of different
antihypertensive drug classes on central aortic pressure. Am J Hypertens.
2004;17(2):118-23.
Jiang XJ, O'Rourke MF, Zhang YQ, He XY, Liu LS. Superior effect of an
angiotensin-converting enzyme inhibitor over a diuretic for reducing aortic
systolic pressure. J Hypertens. 2007;25(5):1095-9.
Schunkert H, Hense HW, Muscholl M, Luchner A, Riegger GA. Association of
angiotensin converting enzyme activity and arterial blood pressure in a
population-based sample. J Hypertens. 1996;14(5):571-5.
Levy D, Anderson KM, Savage DD, Kannel WB, Christiansen JC, Castelli WP.
Echocardiographically detected left ventricular hypertrophy: prevalence and
risk factors. The Framingham Heart Study. Ann Intern Med. 1988;108(1):713.
Messerli FH, Sundgaard-Riise K, Ventura HO, Dunn FG, Oigman W, Frohlich
ED. Clinical and hemodynamic determinants of left ventricular dimensions.
Arch Intern Med. 1984;144(3):477-81.
Dahlof B, Pennert K, Hansson L. Reversal of left ventricular hypertrophy in
hypertensive patients. A metaanalysis of 109 treatment studies. Am J
Hypertens. 1992;5(2):95-110.
Messerli FH, Ketelhut R. Left ventricular hypertrophy: a pressureindependent cardiovascular risk factor. J Cardiovasc Pharmacol. 1993;22
Suppl 1:S7-13.
Nakahara K, Matsushita S, Matsuoka H, Inamatsu T, Nishinaga M, Yonawa M,
Aono T, et al. Insertion/deletion polymorphism in the angiotensinconverting enzyme gene affects heart weight. Circulation. 2000;101(2):14851.
Jeunemaitre X, Lifton RP, Hunt SC, Williams RR, Lalouel JM. Absence of
linkage between the angiotensin converting enzyme locus and human
essential hypertension. Nat Genet. 1992;1(1):72-5.
REFERENCES I 73