Cardiovascular pathology in the year 2000

Cigarette smoking, endothelial injury and cardiovascular

disease

R. MICHAEL PITTILO
Faculty of Health and Social Care Sciences, Kingston University and St George's Hospital
Medical School

Received for publication 19 July 2000

Accepted for publication 24 July 2000

Summary. Despite the fact that the epidemiological evidence linking cigarette
smoking with cardiovascular disease is overwhelming, the precise compo-
nents of cigarette smoke responsible for this relationship and the mechan-
isms by which they exert their effect have not yet been elucidated. There are
however, some promising pointers as a result of recent developments and
this review concentrates on new evidence since earlier reviews of this topic. It
is now known that the endothelium has a vastly more important role than was
ever thought to be the case a decade ago. Its role in health and disease is
increasingly understood, as is the relationship between endothelial injury and
the development of atherosclerosis. There is considerable evidence that
cigarette smoking can result in both morphological and biochemical distur-
bances to the endothelium both in vivo and in cell culture systems. Cigarette
smoke is a complex mixture and only a few components have been exten-
sively studied. Nicotine and carbon monoxide are much less damaging than is
whole smoke. However the free radical components of cigarette smoke have
been shown to cause damage in model systems. Further work will be
necessary to consolidate the evidence base but the data reported in this
review suggest that the free radical components of cigarette smoke may be
responsible for the morphological and functional damage to endothelium that
has been observed in model systems.
Keywords: cigarette smoking, endothelium, injury, nicotine, carbon
monoxide, free radicals, morphology

Epidemiological evidence
Since the ®rst cohort studies indicating that death from
coronary heart disease is more common in smokers than
nonsmokers were published in the 1950s (Doll & Hill
Correspondence: Professor R Michael Pittilo, Faculty of Health
and Social Care Sciences, Kingston University and St George's
Hospital Medical School (University of London), Cranmer
Terrace, London SW17 ORE, UK. Fax: ‡ 44 (0)20 87252159;
E-mail: mpittilo@hcs.sghms.ac.uk
1956; Hammond & Horn 1958), there have been many
reports which convincingly demonstrate that cardiovas-
cular disease is the most important cause of smoking-
related premature death (Fowler 1993). The prospective
investigation of British Doctors commenced in 1951
showed that half the excess mortality from smoking
results from cardiovascular disease with approximately
one third due solely to coronary heart disease (Doll &
Peto 1976). Prospective studies on smokers have shown
that the risk of death from coronary heart disease is

q 2000 Blackwell Science Ltd. Int. J. Exp. Path. (2000), 81, 219±230 219
Cardiovascular pathology in the year 2000
R.M. Pittilo
roughly doubled for both men and women (Fowler 1993)
although there is evidence that the risk is greater for
women (Vriz et al. 1997). Doll (1983) estimated that
cigarette smoking accounted for 25% of the overall
mortality from ischaemic heart disease. More recently
in the United States, Manley (1997) has estimated that
one ®fth of all heart disease-related deaths are due to
cigarette smoking and that smoking, as a single factor,
doubles the risk of heart failure. Cigarette smokers are
also one and a half times more at risk of stroke than
nonsmokers (Fowler 1993). Cigarette smoking is an
independent risk factor in the development of athero-
sclerotic lesions in the internal pudendal and common
penile arteries of young impotent men (Rosen et al.
1991). It is a signi®cant predictor of the presence of
intracranial internal carotid artery atherosclerosis (Ingall
et al. 1991). In the Edinburgh Artery Study, cigarette
smoking was shown to have a direct effect on the risk of
aortic aneurysm which was independent of atherosclero-
sis (Lee et al. 1997). This study also showed that smok-
ing increases the risk of peripheral artery disease more
than heart disease (Fowkes et al. 1992). Perhaps not
surprisingly, cigarette smokers who regularly undertake
physical activity have a reduced cardiovascular mortality
rate than those who have a sedentary existence
(Hedblad et al. 1997). Increased total exposure to
tobacco in humans and a smoking pattern which maxi-
mizes nicotine yield are also associated with an
increased risk of peripheral arterial disease (Powell
et al. 1997).
Endothelial injury
Despite the strong epidemiological evidence that exists
linking cigarette smoking and cardiovascular disease,
the mechanisms by which cigarette smoking causes
disease and the components of smoke responsible
remain poorly understood. Endothelial injury is consid-
ered to be a key initiating event in the pathogenesis of
atherosclerosis (Ross 1986, 1993) and it has therefore
seemed reasonable to hypothesize that cigarette smoke,
or some components of it, may exert its effects by
damaging the endothelium. Components of smoke that
gain access to the circulation will come in contact with
blood and with the vascular endothelial cells that form a
monolayer lining the vessels. These cells are now known
to have a crucial role in controlling the blood circulation
and to be highly active metabolically (Gryglewski et al.
1988; Vane & Botting 1995). Even minor disturbances to
their normal functioning could have signi®cant implica-
tions for the initiation and development of atherosclero-
sis. Endothelial functions, including an increase in
220 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
permeability and decreased nitric oxide (NO) production
along with increased expression of adhesion molecules
and adherence of leukocytes to the vessel wall, have
been shown to be impaired by risk factors for cardiovas-
cular disease such as hypertension, hyperlipidaemia and
hyperglycaemia (Haller 1997). This review examines the
evidence that cigarette smoking can bring about vessel
wall damage with particular reference to the vascular
endothelium.
Damaging effects of cigarette smoking on
human arteries
Direct evidence that cigarette smoking can result in
atherosclerosis continues to be obtained from human
investigations. Using serial quantitative coronary arterio-
graphy, Waters et al. (1996) have shown that cigarette
smoking accelerates coronary progression and new
lesion formation. Interestingly, Lovastatin was shown to
slow the progression of coronary atherosclerosis and
prevent the development of new coronary lesions in
smokers (Waters et al. 1996). The relative thickness of
the intima and media of an artery as measured by
ultrasound can be used to provide an index of athero-
sclerosis. Carotid artery atherosclerosis determined by
ultrasound determination of plaque thickness, a measure
of atherosclerosis, is accelerated depending on the level
of cigarette use and this is independent of age, hyperten-
sion and diabetes (Dempsey & Moore 1992). Cigarette
smoking has an adverse effect on the intimal medial ratio
and stenosis of the carotid artery as measured by ultra-
sound (Howard et al. 1994; Tell et al. 1994; Diezroux et al.
1995). Exposure to environmental tobacco smoke (pas-
sive smoking), and cigarette smoking have been shown
using the intimal/medial ratio as an index to cause
progression of atherosclerosis in the carotid artery
(Howard et al. 1998). Cigarette smoking was shown to
be an independent indicator of the severity of coronary
and thoracic aortic atherosclerosis in men receiving
coronary angiography and transoesophageal echocar-
diography (Inoue et al. 1995). In uraemic patients on
chronic dialysis cigarette smoking is associated with
carotid atherosclerosis as revealed using echo-colour-
Doppler evaluation (Malatino et al. 1999). Studies on the
coronary arteries of trauma victims aged between 15 and
34 years of age suggest that intermediate atherosclerotic
lesions progress rapidly into advanced lesions in
smokers and that intima formerly having early lesions is
replaced by intima with raised lesions (Zieske et al.
1999). This study also demonstrated that smoking was
associated with more extensive fatty streaks and raised
lesions in the abdominal aorta (McGill et al. 1997).

Figure 1. Scanning electron micrograph of control upper
thoracic rat aorta. The endothelial cells have a ¯attened
appearance and the borders can just be discerned. The cell
layer is intact with no exposure of subendothelial collagen ±
´ 1200. Note: Figures 1±7 are scanning electron micrographs
of rat aorta that have been perfusion ®xed. This type of
®xation preserves the vasculature from artefacts such as
shrinkage and contraction.
Radiological observations on women aged between 45
and 64 years showed a direct association between the
development of calci®ed deposits, which have been
shown to represent atherosclerotic change, in the
abdominal aorta and the number of cigarettes smoked
per day over a nine year period (Witteman et al. 1993).
This study suggests that the rate of atherosclerotic
change may be reduced by the cessation of smoking,
but a residual effect appears to be present for a decade
(Witteman et al. 1993). Using ultrasonography to look
at carotid intima and media thickness, the evidence
Figure 2. Scanning electron micrograph from control thoracic
aorta from an animal that has been sham-smoked. The
endothelium is regular and a white blood cell can be seen in
contact with the endothelial surface. The movement of white
blood cells across the endothelium is a normal feature but
they are typically only seen in small numbers ± ´ 1200.
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Cardiovascular pathology in the year 2000
Smoking and endothelial injury
Figure 3. Scanning electron micrograph of the thoracic aorta
from a rat exposed to cigarette smoke. The endothelium is
irregular and some minor projections can be seen protruding
from the cells. These morphological alterations are
accompanied by biochemical disturbances ± ´ 1200.
indicates that atherosclerosis is mediated by compo-
nents of tobacco smoke other than nicotine (Bolinder
et al. 1997).
Cigarette smoking and endothelial
modi®cation
Direct evidence that cigarette smoking could result in
endothelial injury was ®rst obtained from morphological
observations on the umbilical arteries from smoking
mothers. These reports, along with others on the effects
of cigarette smoking on endothelial morphology, have
been previously reviewed by Pittilo (1988, 1990). The
morphological alterations, which included the endothe-
lium having an irregular appearance along with mem-
brane disturbances evidenced by the formation of blebs
or microvillous-like projections, were also associated
with functional changes to the endothelium. In laboratory
rats exposed to cigarette smoke Pittilo et al. (1982) were
able to demonstrate that the morphological alterations to
the endothelium of the thoracic aorta were accompanied
by reduced endothelial prostacyclin production as well
as the adhesion of platelets to apparently intact cells.
Figures 1±7 illustrate the morphological changes asso-
ciated with cigarette smoking. The ability of cigarette
smoking to reduce endothelial prostacyclin production
is well established and the data have been previously
reviewed (Pittilo 1990; Woolf et al. 1993).
Cigarette smoking and the components of
smoke responsible for endothelial injury
Cigarette smoke is a complex mixture and only a few
components have been examined in isolation to assess
221
Cardiovascular pathology in the year 2000
R.M. Pittilo
Figure 4. Scanning electron micrograph of the abdominal
aorta from a rat exposed to cigarette smoke. There is some
minor irregularity of the cells but platelets can be seen
adhering to what may be functionally abnormal, but
nevertheless morphologically intact, endothelium ± ´ 1200.
their effects on endothelial morphology and function. The
effects of nicotine on both the morphology and prosta-
cyclin production of rabbit and cultured human endothe-
lial cells has been reviewed by Bull (1988), Pittilo (1990)
and Woolf et al. (1993), and although deleterious effects
result from exposure, these are much more limited than
those observed with whole smoke (Bull et al. 1985, 1988;
Pittilo et al. 1990). Clinical studies support these ®ndings
in that observations on pipe smokers and users of
transdermal nicotine devices suggest that components of
smoke other than nicotine are the most important causes
of acute cardiovascular events (Benowitz 1997). The
effects of carbon monoxide exposure have also been
reviewed (Pittilo 1988, 1990) and are con¯icting, with
some reports providing evidence of morphological or
functional alterations and others reporting no effects.
Figure 5. Scanning electron micrograph of rat thoracic aorta
following exposure to cigarette smoke. Both a white cell and
platelets can be seen adhering to morphologically abnormal
endothelium ± ´ 1400.
222 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
Figure 6. Scanning electron micrograph of the upper
abdominal aorta from a rat exposed to cigarette smoke. At
lower magni®cation, the endothelium can be seen to be
extremely irregular with a suggestion of apertures between
some raised endothelial cells. Adhering to the surface of the
blood vessel are large numbers of leucocytes. Adhesion of
leucocytes to the endothelium is increased following cigarette
smoking ± ´ 500
Rats exposed to nicotine show an increased frequency
of endothelial cell death which results in enhanced
transendothelial leakage of macromolecules including
low density lipoproteins (Lin et al. 1992). Nicotine has
been shown to enhance the release of platelet-derived
growth factor by bovine aortic smooth muscle cells and is
associated with alterations in the cytoskeleton (Cucina
et al. 2000). Nicotine signi®cantly stimulates DNA synth-
esis and endothelial cell proliferation in bovine pulmonary
artery endothelial cells at concentrations lower than
those obtained in the blood after smoking (Villablanca
1998). In rabbits, administration of nicotine is associated
Figure 7. Scanning electron micrograph of part of the rat
abdominal aorta seen in Figure 6. At this magni®cation the
endothelial cells appear extremely irregular and a very large
number of white blood cells can be seen adhering to the
endothelium ± ´ 1200.

with an acceleration of intimal hyperplasia following
endothelial removal (Hamasaki et al. 1997).
Data derived from epidemiological and animal studies
suggest that carbon monoxide is not atherogenic (Smith
& Steichen 1993). However, exposure of cockerels to
carbon monoxide does not have a discernible effect upon
arteriosclerotic plaque development (Penn et al. 1992).
Bovine pulmonary artery endothelial cells exposed to
carbon monoxide release higher concentrations of nitric
oxide and it has been suggested that carbon monoxide
causes oxidative stress through competition for intra-
cellular binding sites which increase steady state
levels of nitric oxide and the generation of peroxynitrite
by the endothelium (Thom et al. 1997). Carbon
monoxide suppresses the production of endothelin-1
and platelet-derived growth factor B by endothelial cells
(Kourembanas et al. 1997).
There is experimental evidence with cockerels to
indicate that exposure to environmental tobacco smoke
at levels equivalent to those routinely encountered by
people in smoke-®lled environments is suf®cient to pro-
mote arteriosclerotic plaque development (Penn &
Snyder 1993; Penn et al. 1994). In cholesterol-fed
rabbits, passive smoking increases aortic and pulmonary
artery atherosclerosis in a dose-responsive way and is
independent of changes in serum lipids (Zhu et al. 1993;
Sun et al. 1994). One review of the literature concluded
that passive smoking increases platelet activity, accel-
erates atherosclerotic lesion formation, and increases
tissue damage following ischaemia or myocardial infarc-
tion (Glantz & Parmley 1995). However, it has been
argued that the available experimental and epidemiolo-
gical evidence to date does not support an association
between environmental tobacco smoke exposure and an
increased risk of heart disease (Denson 1999; Lee & Roe
1999).
Woolf et al. (1993) and Pittilo & Woolf (1993, 1994)
focused on the possible importance of free radicals as
the mediator of cigarette smoke-induced endothelial
damage for a number of reasons. Cigarette smoke has
been known for many years to be a rich source of free
radicals (Lyons et al. 1958). The scanning electron
microscope changes seen in laboratory rats following
smoke exposure (Pittilo et al. 1982) (Figs 1,2,3,4,5,6,7),
or cultured cells following exposure to plasma from
humans after smoking (Pittilo et al. 1984), were similar
to the changes seen in cardiac myocytes following free
radical-induced lipid peroxidation (Noronha-Dutra &
Steen 1982, 1984). Finally, there is a strong association
between lipid peroxidation and endothelial cell injury
(Hennig & Chow 1988). Woolf et al. (1993) and Pittilo &
Woolf (1994) reviewed some of the supportive evidence
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Cardiovascular pathology in the year 2000
Smoking and endothelial injury
and considered that free radical-induced lipid peroxida-
tion might eventually be shown to be the mechanism
responsible for the epidemiological relationship between
cigarette smoking and atherosclerosis (Pittilo & Woolf
1994). It has been suggested that the high levels of lipid
peroxidation and increased formation of reactive oxygen
species within the vascular wall in atherosclerosis can
overwhelm cellular antioxidant defence mechanisms and
human oxidatively modi®ed LDL-induced expression of
antioxidant stress proteins in vascular cells (Siow et al.
1999).
On the contrary, studies showing no differences in free
radical markers between smokers and nonsmokers sug-
gest that any free radical activity generated from smoking
is adequately scavenged. No signi®cant differences in
free radical markers were found between young adult
smokers and nonsmoking volunteers (Leonard et al.
1995a). Free radical activity due to cigarette smoking
also appears to be adequately scavenged in young
adults with diabetes who are free of signi®cant macro-
vascular disease (Leonard et al. 1995b). Furthermore,
there is evidence to indicate that chemical modi®cation of
glutathione is a major damage mechanism of ®ltered
cigarette smoke with free radical oxidations being less
signi®cant (Maranzana & Mehlhorn 1998).
Despite these ®ndings, there remains strong evidence
to implicate free radicals as an important cause of cigar-
ette smoke-induced endothelial injury. The morphologi-
cal alterations seen with cultured cells exposed to
plasma obtained from volunteers after smoking (Pittilo
et al. 1984) are associated with functional changes to
the endothelium including activation of the hexose-
monophosphate shunt, a sharp increase in the total
glutathione content of the culture medium, release of
angiotensin-converting enzyme from the cells and a
decrease in the ability of the cells to produce ATP, all
of which indicate both oxidative stress and cell injury
(Noronha-Dutra et al. 1993a). Smoking is also a major
determinant of increased plasma-free radical activity in
dyspeptic subjects (Phull et al. 1998).
Free radicals and cigarette smoke
Cigarette smoke consists of two distinct populations
of free radicals; the principal free radical in the tar
phase is a quinone/hydroquinone (Q/QH2) complex
which is an active redox system that can reduce
molecular oxygen to produce superoxide, and in the
gas phase small oxygen-centred and carbon-centred
radicals produced in a steady state by the oxidation of
nitric oxide (NO) to nitrogen dioxide (NO2) react with
reactive species in smoke such as isoprene (Church &
223
Cardiovascular pathology in the year 2000
R.M. Pittilo
Pryor 1985; Pryor et al. 1990). The radicals associated
with the particulate or tar phase of cigarette smoke
are long lived whereas the radicals associated with the
gas or vapour phase have structures that predict a
very short lifespan, although spin trap experiments
demonstrate that these radicals may exist for periods
in excess of ®ve minutes (Church & Pryor 1985; Pryor
et al. 1990). Aqueous extracts of cigarette tar auto-
oxidize to produce semiquinone, hydroxyl, and super-
oxide radicals in air-saturated buffered aqueous
solutions (Zang et al. 1995).
Direct-electron resonance measurements suggest
that superoxide and hydroxl radicals are produced
during the auto-oxidation of hydroquinone and
catechol-related species in aqueous extracts of cigarette
tar (Zang et al. 1995). Fresh cigarette smoke contains
from 300 to 500 parts per million of nitric oxide (Cueto &
Pryor 1994). The nitric oxide radical is produced slowly
from nitric oxide radical donors such as amine com-
plexes, peroxinitrite and other reactants including nitro-
gen oxides (Shinagawa et al. 1998). Nitric oxide can
react with hydrogen peroxide to produce singlet oxygen
which may also be important in mediating cigarette
smoke-induced endothelial damage (Noronha-Dutra
et al. 1993b). Certainly there is evidence to suggest that
nitric oxide in cigarette smoke is important as a mediator
of oxidative damage (Epperlein et al. 1996) and that DNA
damage by cigarette smoke may invoke reactive nitrogen
species (Spencer et al. 1995) as well as reactive oxygen
species (Liu et al. 1999). Analysis of cigarette smoke
demonstrates that the main source of oxygen and hydro-
gen peroxide results from polyphenols in the particulate
phase, and a synergistic effect is observed between
these polyphenols and nicotine (Kodama et al. 1997).
The vapour phase contains a factor which produces
reactive oxygen metabolites from hydrogen peroxide
(Kodama et al. 1997). Free radicals in the gas phase of
cigarette smoke, identi®ed using electron spin resonance
spectrometry, were identi®ed as mainly alkoxyl and alkyl
free radicals with the latter making up approximately two
thirds of the total spectral components (Zhao et al. 1991).
Haemoglobin-impregnated conventional cigarette ®l-
ters are capable of withholding nitric oxide, nitrogen
oxides, hydrogen peroxide, carbon monoxide, alde-
hydes, trace elements and carcinogenic nitrosocom-
pounds from cigarette smoke (Deliconstantinos et al.
1994). This is interesting in view of the fact that the
red blood cells of cigarette smokers contain more glu-
tathione than those of nonsmokers and are more protec-
tive to endothelial cells in culture from hydrogen
peroxide-mediated damage than are the red cells from
nonsmokers (Toth et al. 1986).
224 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
Cigarette smoking and endothelial dysfunction
More recent studies that have not been the subject of
previous reviews continue to provide evidence that cigar-
ette smoke can alter vascular endothelium not only
morphologically but functionally. In recent years, the
discovery of nitric oxide has radically altered our under-
standing of vascular control (Vallance 1997). Both nitric
oxide and carbon monoxide generated within the blood
vessel wall are important cellular messengers involved in
the regulation of vascular smooth muscle tone. (Siow
et al. 1999). Carbon monoxide generated through haeme
oxygenase inhibits mitogen-induced proliferation of
vascular smooth muscle cells and there is evidence
that endogenous carbon monoxide serves as a protec-
tive factor limiting the excessive vascular smooth muscle
cell proliferation associated with vascular disease
(Togane et al. 2000).
Cigarette smoking is associated with dose-related
and potentially reversible impairment of endothelium-
dependent arterial dilation in asymptomatic young adults,
consistent with endothelial dysfunction (Celermajer et al.
1993). It augments endothelial-derived vaso relaxation
but has no effect on endothelium-independent vaso
relaxation (Nene et al. 1997). It leads to a signi®cant
decrease in endothelium-dependent dilatation of the
brachial artery (Lekakis et al. 1997). It causes
immediate constriction of proxymal and distal epicardial
coronary arteries and an increase in coronary vessel
tone despite an increase in myocardial oxygen demand
(Quillen et al. 1993), and cigarette smokers show an
impairment in basal but not stimulated nitric oxide
mediated vaso dilation (McVeigh et al. 1996).
Cigarette smoking has been shown to affect
endothelial nitric oxide synthase activity and protein
levels (Wang et al. 2000). It results in a decrease in
exhaled nitric oxide in humans (Verleden et al. 1999)
suggesting that it may inhibit the enzyme nitric oxide
synthase (Kharitonov et al. 1995). Cigarette smoke
extracts cause an irreversible inhibition of nitric oxide
synthase activity in pulmonary artery endothelial cells
(Su et al. 1998) and a reduction in constitutive nitric
oxide synthase activity in the rat in a dose-dependent
manner (Ma et al. 1999a). Conversely, other studies in
the rat have shown that cigarette smoking results in
an increase in nitric oxide synthase gene expression
and protein production (Wright et al. 1999). However,
lower respiratory tract nitric oxide concentrations are
increased following cigarette smoking (Chambers et al.
1998) although, in humans, plasma and urinary levels
of nitrate, a metabolite of inhaled nitric oxide, have been
shown to be unchanged suggesting that nitric oxide is not

absorbed from the inhaled smoke (Rangemark &
Wennmalm 1996).
Inhibition of nitric oxide synthase activity may explain
the impaired endothelium-independent vasodilation
associated with cigarette smoking (Su et al. 1998). The
decrease in endothelial nitric oxide synthase activity may
also partly explain the high risk of pulmonary and vas-
cular disease in cigarette smokers. Cigarette smoking
affects nitric oxide-mediated coagulation of coronary
artery tone which is associated with a decrease in the
bioactivity of nitric oxide (Kugiyama et al. 1996). It has
been shown to increase isoprostane levels and reduce
the generation of prostacyclin, L-arginine and L-citrulline
in umbilical arteries and veins, and correlates with a
direct vasoconstrictive effect (Obwegeser et al. 1999).
Chronic smoking in the rat leads to age-independent
moderate hypertension and a decrease in penile nitric
oxide synthase activity (Xie et al. 1997).
It has been reported that exposure of cultured bovine
pulmonary artery endothelial cells to carbon monoxide
results in increased release of endothelial nitric oxide
(Thom et al. 1997). The acute cytotoxicity from carbon
monoxide was owing to nitric oxide-derived oxidants
(Thom et al. 1997). Incubation of isolated rabbit aortas
with cigarette smoke extract inhibits endothelium-
dependent relaxation in a dose-dependent manner (Ota
et al. 1997). If this incubation is carried out with free
radical scavengers, attenuation of the inhibition occurs.
This suggests that free radicals in cigarette smoke
extract induce the impairment of endothelium-dependent
relaxation and this may be partly because of the sup-
pression of nitric oxide production (Ota et al. 1997). The
same author has demonstrated, using cultured human
endothelial cells, that cigarette smoke extract sup-
pressed endothelial release of stable metabolites of
nitric oxide and this was attenuated by free radical
scavengers (Ota et al. 1997). The super®cial femoral
veins from rabbits exposed to cigarette smoke have a
signi®cant decrease in endothelium-dependent relaxa-
tion in response to acetylcholine without smooth muscle
injury (Freischlag et al. 1999). Ascorbic acid protects
rabbit arteries from cigarette smoke-induced endothelial
injury by reducing the impairment of endothelium-
dependent acetylcholine relaxation caused by smoking,
probably as a result of oxygen-free radicals (Mays et al.
1999).
It has been suggested that the presence of hypoxia
and exogenous nitric oxide, which lead to endothelial-
dependent and independent vaso-relaxation secondary
to cigarette smoking, may serve to explain the apparent
augmentation of endothelial-derived relaxation in the rat
(Nene et al. 1997). Cigarette smoking has been shown in
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Cardiovascular pathology in the year 2000
Smoking and endothelial injury
the rat to increase aortic endothelial regeneration and
serum levels of nitric oxide following balloon injury of the
thoracic aorta (Sarkar et al. 1999). Acute hypoxia causes
pulmonary vessel constriction, and chronic hypoxia
causes smooth muscle cell replication and extracellular
matrix accumulation, resulting in vessel wall remodelling
(Kourembanas et al. 1997). Oxidized low-density lipo-
protein, hypoxia and pro-in¯ammatory cytokines induce
haem oxygenase expression and activity in vascular
endothelial and smooth muscle cells (Siow et al. 1999).
Cultured human umbilical endothelial cells from
smokers convert signi®cantly more LDL into an athero-
genic form than do cells derived from nonsmokers
(PechAmsellem et al. 1996). The LDL modi®cations
were strongly thiol-dependent and the enhanced super-
oxide production seen in the cells derived from smokers
was dependent on the presence of cysteine in the
medium (PechAmsellem et al. 1996). There is also
evidence that cigarette smoking results in endothelial
dysfunction in hypercholesterolemic patients by enhan-
cing the oxidation of LDL (Heitzer et al. 1996). Cigarette
smoke-modi®ed low-density lipoprotein has been
reported to impair endothelium-dependent relaxation in
isolated rabbit arteries (Kagota et al. 1996).
Cells derived from smokers have higher levels of
intracellular glutathione than those from nonsmokers
and it may be that stimulation of cysteine uptake by the
cells re¯ecting the enhanced total glutathione content
could account for the enhanced superoxide production,
all of which may be relevant to the pathophysiology of
smoking-related cardiovascular disease (PechAmsellem
et al. 1996). It has already been noted that there is
evidence to indicate that chemical modi®cation of
glutathione is a major damage mechanism of ®ltered
cigarette smoke with free radical oxidations being less
signi®cant (Maranzama & Mehlhorn 1998). Endothelial
cell cGMP production is decreased in a dose-dependent
manner following cigarette smoking and endothelial cell
detachment is increased following smoking (Nagy et al.
1997). Externally added thiols protect endothelial cells
from damage and it has been suggested that they may
bind an unknown component of smoke to bring about this
protection (Nagy et al. 1997). In humans, the endothelial
dysfunction observed following cigarette smoking lasts
for about one hour and is not attenuated with repeat
exposure (Lekakis et al. 1998).
Cigarette smoking is associated with increased
monocyte-endothelial cell adhesion in humans (Dovgan
et al. 1994; Adams et al. 1997). Studies on hamsters
using intravital microscopy and scanning electron micro-
scopy demonstrate that cigarette smoking results in
leucocytes adhering in clusters to the aortic endothelium
225
Cardiovascular pathology in the year 2000
R.M. Pittilo
and that aggregates of leukocytes and platelets are
formed (Lehr et al. 1993, 1994). There is evidence that
phagocytes may employ myeloperoxidase-generated
reactive nitrogen intermediates as a physiological path-
way for initiating lipid peroxidation as well as forming
biologically active lipid and sterol oxidation productions in
vivo (Schmitt et al. 1999). Increased generation of
oxygen free radicals by polymorphonuclear leucocytes
may be responsible for the enhanced risk of certain
smoking-related diseases (Kalra et al. 1991). The pro-
motion of neutrophil in®ltration and free radical produc-
tion in rats exposed to cigarette smoke has been shown
to contribute signi®cantly to the development of experi-
mental in¯ammatory bowel disease (Guo et al. 1999).
Cigarette smoking has been shown to increase
re-endothelialization in the rat following large vessel
injury and this is associated with an increase in serum
nitric oxide levels (Sarkar et al. 1999). There is evidence
that cigarette smoke-induced cell proliferation in the
pulmonary arterial vessels is partly mediated through
stimulation of endothelin-A receptors (Dadmanesh &
Wright 1997). Conversely, cigarette smoke represses
angiogenesis in the rat (Ma et al. 1999b). Cigarette
smoking, but not transdermal nicotine delivery, is associ-
ated with borderline increases in plasma endothelin-1
levels but these are restricted to the ®rst 10 minutes after
the onset of smoking (Goerre et al. 1995).
In a study of monozygotic twins discordant for smoking
Calori et al. (1996) found that cigarette smoking was
associated with an atherogenic lipid pro®le along with
changes in platelets and white cells which might re¯ect
endothelial cell damage. Cigarette smoking increases
total serum cholesterol levels (Jorge et al. 1995).
Smokers have mean serum levels that are higher than
nonsmokers for total and low-density lipoprotein choles-
terol and triglyceride whereas high-density lipoprotein
cholesterol levels are lower (Tang et al. 1997). There is
evidence that cigarette smoking results in increased
platelet consumption in human atherosclerotic vessels
as well as the production of larger platelets which are
more active (Kario et al. 1992). Cigarette smoking
causes marked inhibition of substance P-induced tissue
plasminogen activator released in vivo in humans
(Newby et al. 1999). Serum from cigarette smokers
contains higher levels of von Willebrand factor and was
more cytotoxic to endothelial cells in vitro than serum
from nonsmokers (Blann & McCollum 1993).
Conclusions
Since the publication of previous reviews on cigarette
smoking, endothelial injury and cardiovascular disease
226 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
(Pittilo 1988, 1990; Pittilo & Woolf 1993, 1994; Woolf et al.
1993), there have been new reports that demonstrate
that cigarette smoking is associated with blood vessel
wall damage including endothelial injury. Cigarette
smoke is a complex mixture containing a range of
individual components and only a small number of
these have been examined in isolation. Whilst compo-
nents such as nicotine and carbon monoxide have been
the subject of a number of investigations, their damaging
effects in model systems are less than those seen with
whole smoke. Free radicals are an important component
of cigarette smoke and our understanding of their chem-
istry and effects on biological systems has greatly
increased. It seems likely that they will be further impli-
cated as critical to the link between cigarette smoking
and cardiovascular disease.
Acknowledgements
I am grateful to Miss Fatima L. Ramamoorthy for
providing the scanning electron micrographs which
form part of her PhD investigation. I am also indebted
to Mrs D Bunyan for preparing this manuscript.
References
ADAMS M.R., JESSUP W. & CELERMAJER D.S. (1997) Cigarette
smoking is associated with increased human monocyte adhe-
sion to endothelial cells: Reversibility with oral L-arginine but
not vitamin C. J. Am. Coll. Cardio. 29, 491±497.
BENOWITZ N.L. (1997) The role of nicotine in smoking-related
cardiovascular disease. Prev. Med. 26, 412±417.
BLANN A.D. & MCCOLLUM C.N. (1993) Adverse in¯uence of
cigarette smoking on the endothelium. Thromb. Haemost.
70, 707±711.
BOLINDER G., NOREN A., DEFAIRE U. & WAHREN J. (1997) Smoke-
less tobacco use and atherosclerosis: an ultrasonographic
investigation of carotid intima media thickness in healthy
middle-aged men. Atherosclerosis. 132, 95±103.
BULL H.A., PITTILO R.M., BLOW D.J. et al. (1985) The effects of
nicotine on PGI2 production by rat aortic endothelium. Throm.
Haemost. 54, 472±474.
BULL H.A. (1988) The use of cultured endothelial cells in the
study of platelet±vessel wall interactions. In: Platelet±Vessel
Wall Interactions. The Bloomsbury Series in Clinical Science.
(Eds. R.M. PITTILO & S.J. MACHIN) London: Springer-Verlag,
pp. 61±86.
BULL H.A., PITTILO R.M., WOOLF N. & MACHIN S.J. (1988) The
effect of nicotine on human endothelial cell release of
prostaglandins and ultrastruture. Br. J. Exp. Path. 69, 413±
421.
CALORI G., DANGELO A., DELLAVALLE P. et al. (1996) The effect of
cigarette smoking on cardiovascular risk factors: a study of
monozygotic twins discordant for smoking. Thromb. &
Haemost. 75, 14±18.
CELERMAJER D.S., SORENSEN K.E., GEORGAKOPOULOS D. et al.
(1993) Cigarette smoking is associated with dose-related

and potentially reversible impairment of endothelium-
dependent dilation in healthy young adults. Circulation 88,
2149±2155.
CHAMBERS D.C., TUNNICLIFFE W.S. & AYRES J.G. (1998) Acute
inhalation of cigarette smoke increases lower respiratory tract
nitric oxide concentration. Thorax 53, 677±679.
CHURCH D.F. & PRYOR W.A. (1985) Free radical chemistry of
cigarette smoke and its toxicological implications. Environ.
Health Persp. 64, 111±126.
CUCINA A., SAPIENZA P., CORVINO V. et al. (2000) Nicotine induces
platelet-derived growth factor release and cytoskeletal altera-
tion in aortic smooth muscle cells. Surgery 127, 72±78.
CUETO R. & PRYOR W.A. (1994) Cigarette smoke chemistry:
Conversion of nitric oxide to nitrogen dioxide and reactions
of nitrogen oxides with other smoke components as studied by
fourier-transorm-infrared-spectroscopy. Vib. Spectrosc. 7,
97±111.
DADMANESH F. & WRIGHT J.L. (1997) Endothelin-A receptor
antagonist BQ-610 blocks cigarette smoke-induced mitogen-
esis in rat airways and vessels. Am. J. Physiol. Lung Cell. Mol.
Physiol. 16, 614±618.
DELICONSTANTINOS G., VILLIOTOU V. & STRAVRIDES J.C. (1994)
Scavenging effects of haemoglobin and related heme-
containing compounds on nitric oxide, reactive oxidants and
carcinogenic volatile nitrosocompounds of cigarette smoke: a
new method for protection against the dangerous cigarette
constituents. Anticancer. Res. 14, 2727±2726.
DEMPSEY R.J. & MOORE R.W. (1992) Amount of smoking inde-
pendently predicts carotid artery atherosclerosis severity.
Stroke 23, 693±696.
DENSON K.W.E. (1999) Environmental tobacco smoke and
ischaemic heart disease: a critical assessment of recent
meta-analysis and reviews. Med. Sci. Res. 27, 75±82.
DIEZROUX A.V., NIETO F.J., COMSTOCK G.W., HOWARD G. & SZKLO
M. (1995) The relationship of active and passive smoking to
carotid atherosclerosis 12±14 years later. Preven. Med. 24,
48±55.
DOLL R. & HILL A.B. (1956) Lung cancer and other causes of
death in relation to smoking. Brit. Med. J. 4, 1071±1081.
DOLL R. & PETO R. (1976) Mortality in relation to smoking: 20
years' observations on male British doctors. Br. Med. J. 4,
1525±1536.
DOLL R. (1983) Prospects for prevention. Brit. Med. J. 80, 445±
453.
DOVGAN P.S., EDWARDS J.D., ZHAN Z., WILDE M. & AGRAWAL D.K.
(1994) Cigarette smoking increases monocyte adherence to
cultured endothelial cell monolayer. Biochem. Biophys. Res.
Commun. 203, 929±934.
EPPERLEIN M.M., NOUROOZZADEH J., NORONHADUTRA A.A. & WOOLF
N. (1996) Nitric Oxide in cigarette smoke as a mediator of
oxidative damage. Int. J. Experim. Path. 77, 197±200.
FOWKES F.G.R., HOUSLEY E., RIEMERSMA R.A. et al. (1992)
Smoking, lipids, glucose-intolerance, and blood pressure as
risk factors for peripheral atherosclerosis compared with
ischemic heart disease in the Edinburgh Artery Study. Am.
J. Epidemiol. 135, 331±340.
FOWLER G. (1993) Smoking as a risk factor for cardiovascular
disease. In: Cardiovascular disease risk factors and
intervention. (Eds. N. POULTER, P. SEVER S. THOM) Oxford:
Radcliffe Med. Press, pp. 161±169.
FREISCHLAG J.A., JOHNSON D., FAROOQ M.M., et al. (1999)
Cigarette smoke impairs endothelium-dependent relaxation
q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
Cardiovascular pathology in the year 2000
Smoking and endothelial injury
in rabbit super®cial femoral veins. J. Surg. Res. 81, 77±
800.
GLANTZ S.A. & PARMLEY W.W. (1995) Passive smoking and heart
disease: mechanisms and risk. J. Am. Med. Assoc. 273,
1047±1053.
GOERRE S., STAEHLI C., SHAW S. & LUSCHER T.F. (1995) Effect of
cigarette smoking and nicotine on plasma endothelin-1 levels.
J. Cariovasc. Pharmacol. 26, 236±238.
GRYGLEWSKI R.J., BOTTING R.M. & VANE J.R. (1998) Mediators
produced by the endothelial cell. Hypertension 12, 530±548.
GUO X., WANG W.P., KO J.K.S. & CHO C.H. (1999) Involvement of
neutrophils and free radicals in the potentiating effects of
passive cigarette smoking on in¯ammatory bowel disease in
rats. Gastroenterology 117, 884±892.
HALLER H. (1997) Risk factors for cardiovascular disease and the
endothelium. In: Vascular endothelium: physiology, pathol-
ogy, and therapeutic opportunities. (Eds. G. BORN & C.
SCHATTAUR) Stuggart: New Horizon Series 3, Schattauer,
pp. 273±286.
HAMASAKI H., SATO J., MASUDA H., et al. (1997) Effect of nicotine
on the intimal hyperplasia after endothelial removal of rabbit
carotid artery. Gen. Pharmacol. 51, 653±659.
HAMMOND E.C. & HORN D. (1958) Smoking and death rates:
Report on forty-four months of follow-up of 187,783 men. II.
Death rates by cause. JAMA 166, 1294±1308.
HEDBLAD B., OGREN M., ISACSSON S.O. & JANZON L. (1997)
Reduced cardiovascular mortality risk in male smokers who
are physically active: Results from a 25-year follow-up of the
prospective population study men born in 1914. Archiv. Intern.
Med. 157, 893±899.
HEITZER T., YLAHERTTUALA S., LUOMA J. et al. (1996) Cigarette
smoking potentiates endothelial dysfunction of forearm resis-
tance vessels in patients with hypercholesterolemia: Role of
oxidesed LDL. Circulation 93, 1346±1353.
HENNIG B. & CHOW C. (1988) Lipid peroxidation and endothelial
cell injury: implications in atherosclerosis. Free Radic. Biol.
Med. 4, 99±106.
HOWARD G., BURKE G.L., SZKLO M. et al. (1994) Active and
passive smoking are associated with increased carotid wall
thickness: The atherosclerosis risk in communities study.
Archiv. Intern. Med. 154, 1277±1282.
HOWARD G., WAGENKNECHT L.E., BURKE G.L., et al. (1998)
Cigarette smoking and progression of atherosclerosis: The
atherosclerosis risk in communities (ARIC) study. J. Amer.
Med. Assoc. 279, 119±124.
INGALL T.J., HOMER D., BAKER H.L., KOTTKE B.A., OFALLON W.M. &
WHISNANT J.P. (1991) Predictors of intracranial carotid-artery
atherosclerosis: Duration of cigarette smoking and hyperten-
sion are more powerful than serum-lipid levels. Arch. Neurol.
48, 687±691.
INOUE T., OKU K., KIMOTO K. et al. (1995) Relationship of cigarette
smoking to the severity of coronary and thoracic aortic ather-
osclerosis. Cardiology 86, 374±379.
JORGE P.A.R., OZAKI M.R. & ALMEIDA E.A. (1995) Endothelial
dysfunction in coronary vessels and thoracic aorta of rats
exposed to cigarette smoke. Clin. Exp. Pharmacol. Physiol.
22, 410±413.
KAGOTA S., YAMAGUCHI Y., SHINOZUKA K., KWON Y.M. & KUNITOMO M.
(1996) Cigarette smoke-modi®ed low density lipoprotein
impairs endothelium-dependent relaxation in isolated rabbit
arteries. Gen. Pharmacol. 27, 477±481.
KALRA J., CHAUDHARY A.K. & PRASAD K. (1991) Increased production
227
Cardiovascular pathology in the year 2000
R.M. Pittilo
of oxygen free radicals in cigarette smokers. Int. J. Exp.
Pathol. 72, 1±7.
KARIO K., MATSUO T. & MAKAO K. (1992) Cigarette smoking
increases the mean platelet volume in elderly patients with
risk factors for atherosclerosis. Clin. Labor. Haem. 14, 281±
287.
KHARITONOV S.A., ROBBINS R.A., YATES D., KEATING V. & BARNES
P.J. (1995) Acute and chronic effects of cigarette smoking on
exhaled nitric oxide. Am. J. Respir. Crit. Care Med. 152, 609±
612.
KODAMA M., KANEKO M., AIDA M., INOUE F., NAKAYAMA T. & AKIMOTO
H. (1997) Free radical chemistry of cigarette smoke and its
implication in human cancer. Anticancer Res. 17, 433±437.
KOUREMBANAS S., MORITA T., LIU Y.X. & CHRISTOU H. (1997)
Mechanisms by which oxygen regulates gene expression
and cell±cell interaction in the vasculature. Kidney Int. 51,
438±443.
KUGIYAMA K., YASUE H., OHGUSHI M. et al. (1996) De®ciency in
nitric oxide bioactivity in epicardial coronary arteries of
cigarette smokers. J. Am. Coll. Cardiol. 28, 1161±1167.
LEE A.J., FOWKES F.G.R., CARSON M.N., LENG G.C. & ALLEN P.L.
(1997) Smoking, atherosclerosis and risk of abdominal aortic
aneurysm. Eur. Heart J. 18, 671±676.
LEE P.N. & ROE P.J.C. (1999) Environmental tobacco smoke
exposure and heart disease: a critique of the claims of Glantz
and Parmley. Hum. Ecol. Risk Assess. 5, 171±218.
LEHR H.A., KRESS E., MENGER M.D. et al. (1993) Cigarette smoke
elicits leukocyte adhesion to endothelium in hamsters: inhibi-
tion by CUZN-SOD. Free. Rad. Biol. Med. 14, 573±581.
LEHR H.A., FREI B. & ARFORS K.E. (1994) Vitamin C prevents
cigarette smoke-induced leukocyte aggregation and adhesion
to endothelium in-vivo. Proc. Natl. Acad. Sci. U.S.A. 91,
7688±7692.
LEKAKIS J.P., PAPAMICHAEL C.M., VEMMOS C.J. et al. (1997) Effect
of acute cigarette smoking on endothelium-dependent bra-
chial artery dilatation in healthy individuals. Am. J. Cardio. 79,
529±531.
LEKAKIS J.P., PAPAMICHAEL C.M., VEMMOS C.J., STAMATELOPOULOS
K., VOUTSAS A.A. & STAMATELOPOULOS S.F. (1998) Effects of
acute cigarette smoking on endothelium-dependent arterial
dilatation in normal subjects. Am. J. Cardio. 81, 1225.
LEONARD M.B., LAWTON K., WATSON I.D. & MARFARLANE I. (1995a)
Free-radical activity in young adult cigarette smokers. J. Clin.
Pathol. 48, 385±387.
LEONARD M.B., LAWTON K., WATSON I.D., PATRICK A., WALKER A. &
MARFARLANE I. (1995b) Cigarette smoking and free-radical
activity in young adults with insulin dependent diabetes.
Diabetic Med. 12, 46±50.
LIN S.J., HONG C.Y., CHANG M.S., CHIANG B.N. & CHIEN S. (1992)
Long-term nicotine exposure increases aortic endothelial-cell
death and enhances transendothelial macromolecular trans-
port in rats. Arterioscler. Thromb. 11, 1305±1312.
LIU X., LU J.F. & LIU S.J. (1999) Synergistic induction of hydroxyl
radical-induced DNA single-strand breaks by chromium (VI)
compound and cigarette smoke solution. Mutat. Res. Genet
Toxicol. Environ. Mutag. 440, 109±117.
LYONS M.J., GIBSON J.F. & INGRAM D.J.E. (1958) Free radicals
produced in cigarette smoke. Nature 181, 1003±1004.
MCGILL H.C., MCMAHAN C.A., MALCOM G.T., OALMANN M.C. &
STRONG J.P. (1997) Effects of serum lipoproteins and smoking
on atherosclerosis in young men and women. Arterioscl.
Throm. & Vasc. Biol. 17, 95±106.
228 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
MCVEIGH G.E., LEMAY L., MORGAN D. & COHN J.N. (1996) Effects of
long-term cigarette smoking on endothelium-dependent
responses in humans. Am. J. Cardiol. 78, 668±672.
MA L., CHOW J.Y.C. & CHO C.H. (1999a) Cigarette smoking
delays ulcer healing: role of constitutive nitric oxide in
synthase in rat stomach. Am. J. Physiol. Gastrointest. Liver.
Physiol. 39, 238±248.
MA L., CHOW J.Y.C., LIU E.S.L. & CHO C.H. (1999b) Cigarette
smoke and its extracts delays ulcer healing and reduces nitric
oxide synthase activity and angiogenesis in rat stomach. Clin.
Experim. Pharmacol. Physiol. 26, 828±829.
MALATINO L.S., BENEDETTO F.A., MALLAMACI F., TRIPEPI G. &
ZOCCALI C. (1999) Smoking, blood pressure and serum albu-
min are major determinants of carotid atherosclerosis in
dialysis patients. J. Of Nephrol. 12, 256±260.
MANLEY A.F. (1997) Cardiovascular implications of smoking:
The Surgeon General's point of view. J. Health Care Poor
Underserved. 8, 303±310.
MARANZANA A. & MEHLHORN R.J. (1998) Loss of glutathione,
ascorbate recycling, and free radical scavenging in human
erythrocytes exposed to ®ltered cigarette smoke. Arch.
Biochem. Biophys. 350, 169±182.
MAYS B.W., FREISHLAG J.A., EGINTON M.T., CAMBRIA R.A.,
SEABROOK G.R. & TOWNE J.B. (1999) Ascorbic acid prevents
cigarette smoke injury to endothelium-dependent arterial
relaxation. J. Surg. Res. 84, 35±39.
NAGY J., DEMASTER E.G., WITTMANN I., SHULTZ P. & RAIJ L. (1997)
Induction of endothelial cell injury by cigarette smoke.
Endothelium-NY 5, 251±263.
NENE S., GELABERT H., MOORE W., QUINONESBALDRICH W.,
SANTIBANEZGALLERANI A. & IGNARRO L. (1997) Cigarette smoking
increases endothelial-derived vasorelaxation in the rat carotid
artery in a dose-dependent manner. J. Surg. Res. 71, 101±
106.
NEWBY D.E., WRIGHT R.A., LABINJOH C. et al. (1999) Endothelial
dysfunction-impaired endogenous ®brinolysis, and cigarette
smoking: a mechanism for arterial thrombosis and myocardial
infarction. Circulation 99, 1411±1415.
NORONHA-DUTRA A.A. & STEEN E.M. (1982) Lipid peroxidation as a
mechanism of injury in cardiac myocytes. Lab. Invest. 47,
346±353.
NORONHA-DUTRA, A.A., EPPERLEIN M.M. & WOOLF N. (1993a) Effect
of cigarette-smoking on cultured human endothelial-cells.
Cardiovas. Res. 27, 774±778.
NORONHA-DUTRA L.J., EPPERLEIN M.E. & WOOF N. (1993b) Reaction
of nitric oxide with hydrogen peroxide to produce potentially
cytotoxic singlet oxygen as a model for nitric oxide-mediated
killing. FEBS. Lett. 321, 59±62.
NORONHA-DUTRA A.A., STEEN E.M. & WOOLF N. (1984) The early
changes induced by isoproterenol in the endocardium and
adjacent myocardium. Am. J. Pathol. 114, 231±239.
OBWEGESER R., OGUOGHO A., ULM M., BERGHAMMER P. & SINZINGER H.
(1999) Maternal cigarette smoking increases F-2-isoprostanes
and reduces prostacyclin and nitric oxide in umbilical vessels.
Prostagland. Other Lip. Mediat. 57, 269±279.
OTA Y., KUGIYAMA K., SUGIYAMA S. et al. (1997) Impairment of
endothelium-dependent relaxation of rabbit aortas by cigar-
ette smoke extract: Role of free radicals and attenuation by
captopril. Atherosclerosis 131, 195±202.
PENN A., CURRIE J. & SNYDER C. (1992) Inhalation of carbon
monoxide does not accelerate arteriosclerosis in cockerels.
Eur. J. Pharmacol. Environ. Tox. 228, 155±164.

PENN A. & SNYDER C.A. (1993) Inhalation of sidestream cigarette
smoke accelerates development of arteriosclerotic plaques.
Circulation 88, 1820±1825.
PENN A., CHEN L.C. & SNYDER C.A. (1994) Inhalation of steady-
state sidestream smoke from one cigarette promotes arterio-
sclerotic plaque development. Circulation 90, 1363±1367.
PECHAMSELLEM M.A., MYARA I., STOROGENKO M., et al. (1996)
Enhanced modi®cations of low-density lipoproteins (LDL) by
endothelial cells from smokers: a possible mechanism of
smoking-related atherosclerosis. Cardiovasc. Res. 31, 975±
983.
PHULL P.S., PRICE A.B., THORNILEY M.S., GREEN C.J. & JACYNA
M.R. (1998) Plasma free radical activity and antioxidant
vitamin levels in dyspeptic patients: Correlation with smoking
and Helicobacter pylori infection. Eur. J. Gastroenterol.
Hepatol. 10, 573±578.
PITTILO R.M., MACKIE I.J., ROWLES P.M., MACHIN S.J. & WOOLF N.
(1982) Effects of cigarette smoking on the ultrastructure of rat
thoracic aorta and its ability to produce prostacyclin. Throm.
Haemostas. 48, 173±176.
PITTILO R.M., NICHOLSON L.J., CLARKE J.M.F., BLOW C.M. &
WOOLF N. (1984) Cigarette smoke-induced injury of peritoneal
mesothelial cells. Br. J. Exp. Path. 65, 365±370.
PITTILO R.M. (1988) Endothelium and the vessel wall. In: Smok-
ing and Platelet±Vessel Wall Interactions. The Bloomsbury
Series in Clinical Science (Eds. R.M. PITTILO & S.J. MACHIN)
London: Springer-Verlag, pp. 87±101.
PITTILO R.M. (1990) Cigarette smoking and endothelial injury: A
review. Tob. Smok. Atheroscl. 61±78.
PITTILO R.M., BULL. H.A., GULATI S. et al. (1990) Nicotine and
cigarette smoking: effects on the ultrastructure of aortic
endothelium. Int. J. Exp. Path. 71, 573±586.
PITTILO R.M. & WOOLF N. (1993) Cigarette smoking, endothelial
cell injury and atherosclerosis. J. Smoking-Related. Dis. 4,
17±25.
PITTILO R.M. & WOOLF N. (1994) Cigarette smoking as a risk
factor for atherosclerosis. J. Smoking-Related Dis. 5, 43±47.
POWELL J.T., EDWARDS R.J., WORRELL P.C., FRANKS P.J.,
GREENHALGH R.M. & POULTER N.R. (1997) Risk factors asso-
ciated with the development of peripheral arterial disease in
smokers: a case-control study. Atherosclerosis 129, 41±48.
PRYOR W.A., CHURCH D.F., EVANS M.D. et al. (1990) A compar-
ison of the free radical chemistry of tobacco burning cigarettes
that only heat tobacco. Free Radic. Biol. Med. 8, 275±279.
QUILLEN J.E., ROSSEN J.D., OSKARSSON H.J., MINOR R.L., LOPEZ
J.A.G. & WINNIFORD M.D. (1993) Acute effect of cigarette
smoking on the coronary circulation: Constriction of epicardial
and resistance vessels. J. Am. Coll. Cardio. 22, 642±647.
RANGEMARK C. & WENNMALM A. (1996) Smoke-derived nitric oxide
and vascular prostacyclin are unable to counteract the platelet
effect of increased thromoboxane formation in healthy female
smokers. Clin. Physiol. 16, 301±315.
ROSEN M.P., GREENFIELD A.J., WALKER T.G. et al. (1991) Cigarette
smoking: an independent risk factor for atherosclerosis in the
hypogastric-cavernous arterial bed of men with arteriogenic
impotence. J. Urol. 145, 759±763.
ROSS R. (1986) The pathogenesis of atherosclerosis: an update.
N. Engl. J. Med. 314, 488±500.
ROSS R. (1993) The pathogenesis of atherosclerosis: a perspec-
tive for the 1990s. Nature 362, 801±809.
SARKAR R., GELABERT H.A., MOHIUDDIN K.R., THAKOR D.K. &
SANTIBANEZ-GALLERANI A.S. (1999) Effect of cigarette smoke
q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
Cardiovascular pathology in the year 2000
Smoking and endothelial injury
on endothelial regeneration in vivo and nitric oxide levels. J.
Surg. Res. 82, 43±47.
SCHMITT D., SHEN Z.Z., ZHANG R.L. et al. (1999) Leukocytes utilise
myeloperoxidase-generated nitrating intermediates as physio-
logical catalysts for the generation of biologically active oxidised
lipids and sterols in serum. Biochemistry 38, 16904±16915.
SHINAGAWA K., TOKIMOTO T. & SHIRANE K. (1998) Spin trapping of
nitric oxide in aqueous solutions of cigarette smoke. Biochem.
Biophysic. Comm. 253, 99±103.
SIOW R.C.M., SATO H. & MANN G.E. (1999) Heme oxygenase
carbon monoxide signalling pathway in atherosclerosis: anti-
atherogenic actions of bilirubin and carbon monoxide.
Cariovasc. Res. 41, 385±394.
SMITH C.J. & STEICHEN T.J. (1993) The atherogenic potential of
carbon monoxide. Atherosclerosis 99, 137±149.
SPENCER J.P.E., JENNER A., CHIMEL K. et al. (1995) DNA-damage
in human respiratory tract epithelial cells: damage by gas-
phase cigarette smoke apparently involves attack by reactive
nitrogen species in addition to oxygen radicals. FEBS Lett.
375, 179±182.
SU Y.C., HAN W.H., DE GIRALDO C., LI Y. & BLOCK E.R. (1998)
Effect of cigarette smoke extract on nitric oxide synthase in
pulmonary artery endothelial cells. Am. J. Resp. Cell Mol. Biol.
19, 819±825.
SUN Y.P., ZHU B.Q., SIEVERS R.E., GLANTZ S.A. & PARMLEY W.W.
(1994) Metoprolol does not attenuate atherosclerosis in lipid-
fed rabbits exposed to environmental tobacco smoke. Circu-
lation 89, 2260±2265.
TANG J., MUIR J., LANCASTER T., JONES L. & FOWLER G. (1997)
Health pro®les of current and former smokers and lifelong
abstainers. J. Roy. Coll. Physic. London 31, 304±309.
TELL G.S., POLAK J.F., WARD B.J., KITTNER S.J., SAVAGE P.J. &
ROBBINS J. (1994) Relation of smoking with carotid artery wall
thickness and stenosis in older adults: The cardiovascular
health study. Circulation. 90, 2905±2908.
THOM S.R., XU Y.A. & ISCHIROPOULOS H. (1997) Vascular endo-
thelial cells generate peroxynitrite in response to carbon
monoxide exposure. Chem. Res. Toxicol. 10, 1023±1031.
TOGANE Y., MORITA T., SUEMATSU M., ISHIMURA Y., YAMAZAKI J. &
KATAYAMA S. (2000) Protective roles of endogenous-carbon
monoxide in neointimal development elicited by arterial injury.
Am. J. Physiol. Heart Circul. Physiol. 278, 623±632.
TOTH K.M., BERGER E.M., BEEHLER C.J. & REPINE J.E. (1986)
Erythrocytes from cigarette smokers contain more glutathione
and catalase and protect endothelial cells from hydrogen
peroxide better than do erythrocytes from non-smokers.
Amer. Lung Assoc. New York, 134, 281±284.
VALLANCE P. (1997) Exploring vascular nitric oxide in health
and disease: The Goulstonian Lecture (1996). J. Roy. Coll.
Physic. Lon. 31, 321±327.
VANE J.R. & BOTTING R.M. (1995) Orchestration of the blood
circulation by the endothelium. In: The endothelial cell in
health and disease. (Eds. J.R. VANE, G. BORN & D. WELZEL)
Stuggart: Schattauer, pp. 1±32.
VERLEDEN G.M., DUPONT L.J., VERPEUT A.C. & DEMEDTS M.G.
(1999) The effect of cigarette smoking on exhaled nitric
oxide in mold steroid-naõÈve asthmatics. Chest 116, 59±64.
VILLABLANCA A.C. (1998) Nicotine stimulates DNA synthesis and
proliferation in vascular endothelial cells in vitro. J. Appl.
Physiol. 84, 2089±2098.
VRIZ O., NESBITT S., KRAUSE L., MAJAHALME S., LU H. & JULIUS S.
(1997) Smoking is associated with higher cardiovascular risk
229
Cardiovascular pathology in the year 2000
R.M. Pittilo
in young women than in men: The Tecumseh Blood Pressure
Study. J. Hypertens. 15, 127±134.
WANG X.L., SIM A.S., WANG M.X., MURRELL G.A.C., TRUDINGER B.
& WANG J. (2000) Genotype dependent and cigarette speci®c
effects on endothelial nitric oxide synthase gene expression
and enzyme activity. Febs Lett 471, 45±50.
WATERS D., LESPERANCE J., GLADSTONE P. et al. (1996) Effects of
cigarette smoking on the angiographic evolution of coronary
atherosclerosis: a Canadian Coronary Atherosclerosis Inter-
vention Trial (CCAIT) substudy. Circulation 94 614±621.
WITTEMAN J.C.M., GROBBEE D.E., VALKENBURGH H.A., VANHERMERT
A.M., STIJNEN T. & HOFMAN A. (1993) Cigarette smoking and
the development and progression of aortic atherosclerosis: a
9-year population-based follow-up-study in women. Circula-
tion 88, 2156±2162.
WOOLF N., PITTILO R.M., ROWLES P.M., NORONHA-DUTRA A.A. &
EPPERLEIN M.M. (1993) Cigarette smoking and atherosclerosis.
In: Cardiovascular Disease: Risk Factors and Intervention.
(Eds. N. POULTER, P. SEVER & S. THOM) Oxford: Radcliffe Med.
Press. pp. 171±183.
230 q 2000 Blackwell Science Ltd, International Journal of Experimental Pathology, 81, 219±230
WRIGHT J.L., DAI J., ZAY K., PRICE K., GILKS C.B. & CHURG A.
(1999) Effects of cigarette smoke on nitric oxide synthase
expression in the rat lung. Lab. Investig. 79, 975±983.
XIE Y.N., GARGAN H., NG C., RAJFER J. & GONZALEZCADAVID N.F.
(1997) Effect of long-term passive smoking on erectile func-
tion and penile nitric oxide synthase in the rat. J. Urol. 157,
1121±1126.
ZANG L.Y., STONE K. & PRYOR W.A. (1995) Detection of free
radicals in aqueous extracts of cigarette tar by electron-spin
resonance. Free Radic. Biol. Med. 19, 161±167.
ZHAO B.L., YAN L.J., HOU J.W. & XIN W.J. (1991) ESR spin
trapping studies on the free radicals in gas phase of cigarette
smoke. Chin. Med. J. 104, 591±594.
ZHU B.Q., SUN Y.P., SIEVERS R.E., ISENBERG W.M., GLANTZ S.A. &
PARMLEY W.W. (1993) Passive smoking increases experimen-
tal atherosclerosis in cholesterol-fed rabbits. J. Amer. Coll.
Cardio. 21, 225±232.
ZIESKE A.W., TAKEI H., FALLON K.B. & STRONG J.P. (1999)
Smoking and atherosclerosis in youth. Atherosclerosis 144,
403±408.