dilute diesel exhaust, (ii) pure carbon nanoparticulate, (iii) filtered diesel exhaust, or (iv) filtered air, in a randomized
double blind cross-over study. Following each exposure, forearm blood flow was measured during intra-brachial
bradykinin, acetylcholine, sodium nitroprusside, and verapamil infusions. Compared with filtered air, inhalation of
diesel exhaust increased systolic blood pressure (145+4 vs. 133+3 mmHg, P , 0.05) and attenuated vasodilatation
to bradykinin (P ¼ 0.005), acetylcholine (P ¼ 0.008), and sodium nitroprusside (P , 0.001). Exposure to pure carbon
nanoparticulate or filtered exhaust had no effect on endothelium-dependent or -independent vasodilatation. To
determine the direct vascular effects of nanoparticulate, isolated rat aortic rings (n ¼ 6–9 per group) were assessed
in vitro by wire myography and exposed to diesel exhaust particulate, pure carbon nanoparticulate and vehicle. Com-
pared with vehicle, diesel exhaust particulate (but not pure carbon nanoparticulate) attenuated both acetylcholine
(P , 0.001) and sodium-nitroprusside (P ¼ 0.019)-induced vasorelaxation. These effects were partially attributable
to both soluble and insoluble components of the particulate.
Combustion-derived nanoparticulate appears to predominately mediate the adverse vascular effects of diesel exhaust
inhalation. This provides a rationale for testing environmental health interventions targeted at reducing traffic-derived
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Air pollution † Diesel exhaust † Nanoparticles † Endothelium † Blood flow
Prevention and epidemiology
Combustion-derived nanoparticulate induces
the adverse vascular effects of diesel exhaust
Nicholas L.Mills1†, Mark R.Miller1*†, Andrew J.Lucking1, JonBeveridge1, LauraFlint1,
A. John F.Boere2, Paul H.Fokkens2, Nicholas A.Boon1, ThomasSandstrom3,
AndersBlomberg3, RodgerDuffin4, KenDonaldson4, Patrick W.F.Hadoke1,
Flemming R.Cassee2‡, and David E.Newby1‡
1The BHF/University Centre for Cardiovascular Science, Edinburgh University, The Queens Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK;
2National Institute for Public Health and the Environment, Bilthoven, The Netherlands;3Department of Respiratory Medicine and Allergy, Umea ˚ University, Umea, Sweden;
and4ELEGI Colt Laboratory, Centre for Inflammation Research, Edinburgh University, Edinburgh, UK
Received 5 February 2011; revised 4 April 2011; accepted 25 May 2011; online publish-ahead-of-print 13 July 2011
See page 2613 for the editorial comment on this article (doi:10.1093/eurheartj/ehr200)
Exposure to road traffic and air pollution may be a trigger of acute myocardial infarction, but the individual pollutants
responsible for this effect have not been established. We assess the role of combustion-derived-nanoparticles in
mediating the adverse cardiovascular effects of air pollution.
To determine the in vivo effects of inhalation of diesel exhaust components, 16 healthy volunteers were exposed to (i)
†Both authors contributed to equal first authorship.
* Corresponding author. Tel: +44 131 242 9334, Fax: +44 131 242 9215, Email: firstname.lastname@example.org
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: email@example.com
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article
for non-commercial purposes provided that the original authorship is properly and fully attributed; the Journal, Learned Society and Oxford University Press are attributed as the
original place of publication with correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this
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‡Both authors are equal senior authors.
European Heart Journal (2011) 32, 2660–2671
Air pollution is increasingly recognized as an important and mod-
ifiable risk factor for cardiovascular disease.1Acute exposure has
been linked to a range of adverse cardiovascular events, including
hospital admissions with angina,2myocardial infarction,3and
heart failure,4and long-term exposure increases the lifetime risk
of death from coronary heart disease.5Long-term residential
exposure to air pollution is also associated with the extent of
atherosclerosis in the carotid and coronary blood vessels.6–12
These associations are strongest for fine particulate matter
(PM2.5) air pollution that arises from a variety of sources, including
the combustion of diesel fuel by automobiles. This important
source of PM2.5is thought to explain the association between tran-
sient exposure to road traffic and the triggering of acute myocar-
We have previously demonstrated that inhalation of dilute diesel
exhaust impairs vascular function,14and has pro-thrombotic effects
in both healthy volunteers15and patients with coronary heart
disease.16Diesel exhaust is a complex mixture of gases, particles,
and volatiles, and our earlier studies preclude identification of
the components responsible for these adverse effects. The
recent American Heart Association statement highlights the need
for a greater understanding of the role of these different com-
ponents in mediating the cardiovascular effects of air pollution.1
Ultrafine particles, nitrogen dioxide, carbon monoxide, sulphur
dioxide, and organics chemicals may all play a role. Transition
metals and organic constituents, such as polyaromatic hydrocar-
bons, on the surface of particles are believed to be key mediators
of the harmful actions of diesel exhaust.17Understanding the role
of individual pollutants is an important consideration in the science
of emission control technology and is essential to guide public
health and environmental policy.
Using complementary clinical and pre-clinical studies, our aim
was to establish the role of combustion-derived particles in deter-
mining the adverse vascular effects of diesel exhaust inhalation.
Using a specially designed human exposure chamber and particle
filtration system, we compared the effects of dilute diesel
exhaust with the gaseous components alone, and with ‘clean’
pure carbon nanoparticles. The direct vascular effects of diesel
exhaust particles and carbon nanoparticles were compared in iso-
lated rat aortic rings using myography.
Sixteen healthy male non-smokers aged between 18 and 32 years par-
ticipated in these studies that were performed with the approval of the
local research Ethics Committee, in accordance with the Declaration
of Helsinki, and the written informed consent of all volunteers. Sub-
jects taking regular medication and those with clinical evidence of
atherosclerotic vascular disease, arrhythmia, diabetes mellitus, hyper-
tension, renal or hepatic failure, asthma, significant occupational
exposure to air pollution, or an inter-current illness likely to be associ-
ated with inflammation were excluded from the study. Subjects had
normal lung function and reported no symptoms of respiratory tract
infection for at least 6 weeks prior to or during the study. Routine
measures of clinical haematology and biochemistry, including blood
glucose, were normal.
Subjects attended on four separate occasions at least 2 weeks apart
and receive a double-blind randomized cross-over exposure to fil-
tered air, carbon nanoparticles, diesel exhaust and filtered diesel
exhaust from which the particulate phase was removed. All subjects
were subjected to all four exposures with at least 2-week washout
between exposures. The order of the exposures was selected using
a random allocation taken from a balanced block of 24 to account
for all permutations. On each occasion, each subject was exposed
for 2 h in a specially built exposure chamber. During each exposure,
they performed moderate exercise (minute ventilation 25 L/min/m2)
on a bicycle ergometer that was alternated with rest at 15-min
Based on previous exposure studies,14vascular assessments were
performed 6–8 h following each exposure. All subjects abstained
from alcohol for 24 h and from food, tobacco, and caffeine-containing
drinks for at least 4 h before each vascular study. Studies were carried
out in a quiet, temperature-controlled room maintained at 22–248C,
with subjects lying supine. All subjects remained indoors between
the exposure and vascular assessment to minimize additional exposure
to particulate air pollution.
All exposures were delivered in a purpose-built exposure chamber
(Figure 1). The air in the exposure chamber inlet was continuously
monitored for nitrogen oxides [chemiluminescence NO-NO2-NOx
analyser, model 40W, Thermo Environmental Instruments (TEI),
USA], carbon monoxide (gas filter correlation CO analyser, model
48, TEI, USA), sulphur dioxide (pulsed fluorescence SO2analyser,
TEI, USA), and ozone (photometric analyser, monitor labs 9810O3,
Measurement Controls Corporation, USA). Particle number was
determined using a condensation particle counter (Model 3022A,
Thermo Systems Incorporated, USA). Particle mass was continuously
monitored using a DataRam nephelometer (Measuring Instruments
for the Environment corporation, USA) to standardize diesel
exposures, with the precise mass determined by gravimetric filter
measurements (teflon 2.0 mm 4.7 mm, PALL Life Sciences, USA).
Temperature and humidity in the chamber were measured with a ther-
mometer and hygrometer (ETHG889, Oregon Scientific, Portland OR,
Diesel exhaust was generated from an unloaded diesel engine
(Deutz, 4 cylinder, 2.2 L, 500 rpm) using gas oil (Petroplus Refining
Teesside Ltd, UK). More than 90% of the exhaust was shunted away,
and the remaining part was diluted with air, passed through an impac-
tor with a cutoff of 0.1 mm, and fed at 75 L/min into the exposure
chamber at steady-state concentration of ?300 mg/m3. Filtered
exhaust was generated in an identical manner, but the exhaust was
passed through a highly efficient TE38 Teflon filter (Schleicher &
Schuell, Dassel, Germany) to remove particles before being fed into
An aerosol of carbon nanoparticles was generated from graphite
electrodes by an electric spark discharge generator (Palas CFG1000,
Palas GmbH, Karlsruhe, Germany) in an atmosphere of argon. The
output of the generator was mixed with filtered air, passed through
an impactor with a cutoff of 0.1 mm, and fed at 75 L/min into the
exposure chamber at steady-state concentration. The number concen-
tration in the exposure chamber was maintained at 4000 × 103
Nanoparticulate and vascular dysfunction
particles/cm3as this was associated with the maximum achievable mass
All subjects underwent brachial artery cannulation with a 27-standard
wire gauge steel needle under controlled conditions. Following a
30-min baseline saline infusion, acetylcholine at 5, 10, and 20 mg/min
[endothelium-dependent vasodilator that does not release tissue plas-
minogen activator (t-PA); Merck Biosciences, Switzerland], bradykinin
at 100, 300, and 1000 pmol/min (endothelium-dependent vasodilator
that releases t-PA; Merck Biosciences, Switzerland), and sodium nitro-
prusside at 2, 4, and 8 mg/min (endothelium-independent vasodilator
that does not release t-PA; David Bull Laboratories, UK) were
infused for 6 min at each dose. The three vasodilators were separated
by 20-min saline infusions and given in a randomized order. Verapamil
at 10, 30, and 100 mg/min (endothelium and nitric oxide-independent
vasodilator that does not release t-PA) was infused at the end of the
Forearm blood flow was measured in the infused and non-infused
arms by venous occlusion plethysmography using mercury-in-silastic
strain gauges as described previously.20Supine heart rate and blood
pressure in the non-infused arm were monitored at intervals through-
out each study using a semi-automated non-invasive oscillometric
Venous cannulae (17-gauge) were inserted into large subcutaneous
veins of the ante-cubital fossae of both arms. Blood (10 mL) was with-
drawn simultaneously from each arm at baseline and during the infu-
sion of each dose of bradykinin, and collected into acidified buffered
citrate (Stabilyte tubes, Biopool International) for t-PA assays, and
citrate (BD Vacutainer) for plasminogen activator inhibitor type 1
(PAI-1) assays. Samples were kept on ice before being centrifuged at
2000 g for 30 min at 48C. Platelet-free plasma was decanted and
stored at 2808C before assay. Plasma t-PA and PAI-1 antigen and
activity concentrations were determined by enzyme-linked immuno-
sorbant assays (t-PA Combi Actibind Elisa Kit, Technoclone, Vienna,
Austria and Elitest PAI-1 antigen and Zymutest PAI-1 Activity,
Hyphen Biomed, Neuville-Sur-Oise, France). Haematocrit was deter-
mined by capillary tube centrifugation at baseline and during infusion
of bradykinin 1000 pmol/min.
Blood samples were taken immediately before, 2, 6, and 24 h after
the exposure and analysed for total cells, differential count, and
platelets using an autoanalyzer.
Preparation of particle suspensions
Suspensions of diesel exhaust particles and vascular tissues were per-
formed as described previously.18Briefly, diesel exhaust particles
(SRM-2975; National Institute of Standards and Technology, Gaithers-
burg, USA) and carbon nanoparticles (Printex 90; Degussa, Frankfurt,
Germany) were suspended in Krebs buffer at a stock concentration
of 2 mg/mL, followed by vortexing and sonication.
The components of diesel exhaust particles were separated by serial
washes in Krebs buffer (to remove constituents soluble in aqueous
solutions) or dichloromethane (to remove organic constituents). For
aqueous extraction, a suspension of diesel exhaust particles was pre-
pared at a concentration of 2 mg/mL in Krebs buffer and agitated over-
night at room temperature. The suspension was then centrifuged at
15 800 g for 10 min and the aqueous supernatant separated from
the particle pellet. The pellet was resuspended in an identical
volume of Krebs buffer and both solutions were recentrifuged and sep-
arated twice more to remove contaminants. The aqueous extract of
Figure 1 Exposure chamber and particle filtration system for clinical studies. Diesel exhaust was generated from an unloaded diesel engine
using gas oil. More than 90% of the exhaust was shunted away, and the remaining part was diluted with air and fed at 75 L/min into the exposure
chamber at steady-state concentration. Diesel exhaust particulate was removed for the control filtered exhaust exposure by passing dilute
exhaust through a teflon filter. Carbon nanoparticles were generated using a Palas GFG 1000 spark discharge generator. NP, nanoparticles;
T, temperature; RH, relative humidity; CO, carbon monoxide; SO2, sulphur dioxide; NOx, nitrogen oxides; CPC, condensation particle
counter; SMPS, scanning mobility particle sizer.
N.L. Mills et al.
diesel exhaust particles was passed through a syringe filter (0.22 mm;
Millipore, Fisher Scientific, Loughborough, UK). Krebs buffer was
added to the particle pellet to provide at a concentration of 2 mg/mL
and resonicated for 15 min to ensure complete resuspension and
disaggregation of the aqueous-washed diesel exhaust particles.
The aromatic organic constituents on the surface of diesel exhaust
particles were separated through modification of the method used
by Koike and Kobayashi.19Briefly, particles were suspended in dichlor-
omethane at 10 mg/mL in Krebs buffer and agitated for .24 h at room
temperature. Suspensions were processed as for the aqueous separ-
ation; following three centrifugations, dichloromethane was completely
evaporated (378C for 4 h) from the particulate pellet and extract. The
particulate pellet was resuspended at 2 mg/mL in Krebs buffer after
15 min of sonication (organic-washed diesel exhaust particles), and
the residue from the evaporated extract was resuspended in dimeth-
lysulphide at a concentration equivalent to that present in the original
10 mg/mL stock (organic extract from diesel exhaust particles). The
final concentration of dimethyl sulphoxide in the biological assays
was 1% and preliminary experiments showed that this concentration
did affect vascular responses.
All experiments were performed according to the Animals (Scientific
Procedures) Act 1986 (UK Home Office). Rings of thoracic aorta
from adult male Wistar rats (n ¼ 48 in total; 6–9 per experimental
group) were mounted in a multi-myograph system (610M; Danish
Myo Technology, Aarhus, Denmark) under a baseline tension of
14.7 mN. Vessels were pre-treated with diesel exhaust particles
(100 mg/mL) or the aqueous and organic extracts from diesel
exhaust particles, and carbon nanoparticles (100 mg/mL) 20 min
prior to, and throughout, generation of concentration–response
curves. Concentration–response curves to phenylephrine (1 nM–
10 mM) were obtained, and a concentration that produced 80%
maximum contraction (EC80; 0.1–1 mM) was chosen for each
individual rat aortic ring. Following contraction, cumulative concen-
tration–response curves were obtained for the endothelium-
dependent vasodilator acetylcholine
endothelium-independent nitric oxide donor sodium nitroprusside
(0.1 nM–1 mM). In some experiments rings were incubated with the
superoxide scavenging enzyme—superoxide dismutase (SOD; 100 U/
mL)—or the hydroxyl radical scavenger—mannitol (5 mM)—alone
or via co-incubated with particles (10 or 100 mg/mL) 20 min prior to
(1 nM–10 mM) andthe
Data analysis and statistics
Based on our previous studies of endothelial vasomotor function and
endogenous fibrinolysis, to detect a 20% difference in forearm blood
flow and a 16% difference in t-PA release, we require sample sizes
of n ¼ 16 at 80% power and two-sided P , 0.05.
Plethysmographic data were analysed as described previously.20Esti-
mated net release of t-PA antigen and activity was defined as the
product of the infused forearm plasma flow (based on the mean hae-
matocrit and the infused forearm blood flow) and the concentration
difference between the infused and non-infused arms. Data from the
myography studies were analysed as described previously,18using
two-way analysis of variance (ANOVA) to compare concentration–
response curves and unpaired t-test to compare differences in
maximum contraction. Vasodilator responses were expressed as a per-
centage of the pre-contraction to EC80phenylephrine, where 100%
relaxation represents a complete abolition of phenylephrine-induced
tone. Continuous variables are reported as mean+SEM. Statistical
Systemic effects of exposure to filtered air, diesel exhaust, and carbon nanoparticles
P ¼ 0.020
P ¼ 0.640
P , 0.001
P ¼ 0.292
P ¼ 0.019
P ¼ 0.076
P ¼ 0.928
P ¼ 0.062
t-PA antigen, ng/mL
P ¼ 0.010
P ¼ 0.394
t-PA activity, ng/mL
P , 0.001
P ¼ 0.083
PAI-1 antigen, ng/mL
P , 0.001
P ¼ 0.782
PAI-1 activity, ng/mL
P , 0.001
P ¼ 0.316
Values are reported as mean+standard deviation (n ¼ 16); ANOVA with repeated measures.
Nanoparticulate and vascular dysfunction
analyses were performed with GraphPad Prism (Graphpad Software,
Inc., La Jolla, CA, USA) using ANOVA with repeated measures with
Bonferroni post hoc tests of selected comparisons where appropriate.
Statistical significance was taken at two-sided P , 0.05.
All studies were well tolerated with no adverse effects. Total leu-
cocytes and neutrophils were increased 2 and 6 h following
exposures (P , 0.001), but this was unaffected by the type of
exposure (Table 1). Plasma concentrations of t-PA antigen and
activity as well as PAI-1 antigen and activity also displayed circadian
variation, but were not affected by exposure to diesel exhaust or
pure carbon nanoparticles.
Particulate mass and number concentrations in dilute diesel
exhaust were similar to our previous exposures14,15at 348+
16 mg/m3and 1198 × 103particles/cm3, respectively (Table 2). Fil-
tration reduced the number of particles by 1000-fold (2 × 103par-
ticles/cm3), resulting in a 60-fold lower mass concentrations of 6+
4 mg/m3. Gaseous pollutants were not affected by particle filtration
and neither temperature nor humidity varied between exposures.
Despite delivering four-fold more carbon nanoparticles (3865 ×
103vs. 1198 × 103particles/cm3) using the Palas generator, the
mass concentration of particulate was only 70+7 mg/m3. The
difference in mass concentration between carbon and diesel
exhaust exposures was due to the smaller size of carbon nanopar-
ticles (median diameter 37+1 vs. 67+1 nm, respectively).
Greater than 85% of diesel exhaust particles and 95% of carbon
particles, by particle number, had an aerodynamic diameter of
There were no differences in resting heart rate or basal forearm
blood flow between exposures (Table 3). Inhalation of dilute
diesel and filtered diesel exhaust increased systolic blood pressure
(145+4 and 144+3 mmHg, respectively) compared with
filtered air (133+3 mmHg; P ¼ 0.012). While there was a
dose-dependent increase in blood flow with each vasodilator
(P , 0.001 for all), this response was attenuated during bradykinin
(P ¼ 0.005), acetylcholine (P ¼ 0.008), and sodium nitroprusside
(P , 0.001) infusions following exposure to diesel exhaust com-
pared with filtered air (Figure 2). Verapamil-induced vasodilatation
PM (teflon filter), mg/m3
Particle number, ×1000/cm3
Particle diameter, nm–
Carbon monoxide, p.p.m.0.2+0.4
Sulphur dioxide, p.p.m.0.1+0.0
Nitric oxide [NO], p.p.m.
Nitrogen dioxide [NO2], p.p.m.
NOx[NO + NO2], p.p.m.
Relative humidity,% 72+8
Characterization of exposure conditions
Filtered air Diesel exhaustFiltered exhaustCarbon
Values are presented as number or mean+standard deviation (n ¼ 16).
Heart rate, b.p.m. 68+8 67+12
Systolic blood pressure, mmHg133+12 145+16*
Diastolic blood pressure, mmHg69+8 69+8
Infused FBF, mL/100 mL tissue/min 2.4+0.82.1+0.8
Non-infused FBF, mL/100 mL tissue/min 1.7+0.8 1.9+0.8
Baseline haemodynamic variables from vascular assessment at 6 h
Filtered air Diesel exhaust Filtered exhaustCarbon Significance
P ¼ 0.475
P ¼ 0.012
P ¼ 0.904
P ¼ 0.686
P ¼ 0.410
Values are reported as mean+standard deviation. ANOVA with repeated measures. Bonferroni post test. FBF, forearm blood flow.
*P , 0.05 exposure vs. filtered air.
N.L. Mills et al.
was not significantly impaired following exposure to diesel exhaust
(P ¼ 0.08). Neither exposure to filtered diesel exhaust (Figure 3)
nor pure carbon nanoparticles (data not shown) had any effect
on endothelium-dependent and -independent vasodilatation. Bra-
dykinin caused a dose-dependent increase in plasma t-PA antigen
and activity concentrations (P , 0.001) that was not altered by
any of the exposures compared with filtered air (data not shown).
In vitro exposure of rat aortic rings to diesel exhaust particles pro-
duced a small increase in the maximum contractile response to
phenylephrine (P ¼ 0.03, unpaired t-test; Figure 4A), without affect-
ing the sensitivity to phenylephrine. Pure carbon nanoparticles did
not significantly alter responses to phenylephrine and, overall,
both particles had no significant effect on concentration–response
curves to phenylephrine when expressed as a percentage of
maximum contraction (P ¼ 0.64 and P ¼ 0.86, respectively;
exhaust particles (P , 0.001; Figure 4C). Although there was an
apparent rightward shift in the concentration–response curve to
acetylcholine with carbon nanoparticles, vasorelaxation was not
significantly affected (P ¼ 0.139; Figure 4C). Diesel exhaust particles
caused a small but significant inhibition of relaxation to sodium
nitroprusside (P ¼ 0.019) compared with control (Figure 4D),
whereas carbon nanoparticles did not (P ¼ 0.224).
Following separation of the aqueous soluble constituents from
diesel exhaust particulate matter, both washed particles and
Figure 2 Forearm blood flow 6–8 h after exposure to diesel exhaust or air. Infused (solid line) and non-infused (dashed line) forearm blood
flow in healthy subjects, 6–8 h following diesel (filled circle) or air (open circle) exposure, during intra-brachial infusion of bradykinin, acetyl-
choline, sodium nitroprusside, or verapamil: for all dose responses P , 0.0001. For diesel exposure (filled circle) vs. air (open circle); bradykinin
(**P ¼ 0.005), acetylcholine (**P ¼ 0.008), sodium nitroprusside (***P , 0.001), and verapamil (P ¼ 0.08).
Nanoparticulate and vascular dysfunction
solution were able to inhibit vasodilator responses to acetylcholine
(P , 0.01 for both), with the combined effects of both constituents
beingequal tothat ofuntreated
Dichloromethane-washed diesel exhaust particles markedly inhib-
ited acetylcholine-induced vasodilatation (P ¼ 0.010); the magni-
tude of which was not statistically different from untreated diesel
exhaust particles (P ¼ 0.103; Figure 4F). Accordingly, the organic
extract of diesel exhaust particles removed by dichloromethane
had no effect on relaxation to acetylcholine (P ¼ 0.645).
The superoxide free radical scavenger, superoxide dismutase
(SOD), partially reversed the inhibitory effects of diesel exhaust
particles on acetylcholine responses (P , 0.001; Figure 5A). Super-
oxide dismutase completely reversed the action of Krebs-washed
diesel particles (P ¼ 0.03), the Krebs extract (P ¼ 0.03), and
dichloromethane-washed diesel particles (P ¼ 0.009), as well as
the response to carbon nanoparticles (P ¼ 0.18), although this
effect was not statistically significant due to the modest inhibitory
effect of these particles alone (Figure 5B–E). The hydroxyl radical
scavenger, mannitol, however, had no effect on the action of
diesel particles (10 mg/mL) on acetylcholine responses (P ¼ 0.84;
attempted to identify the components responsible for the adverse
cardiovascular effects of air pollution. When assessed in the clinic,
we showed that the vascular dysfunction associated with diesel
exhaust inhalation is prevented by particle filtration. However, the
nature of the particles appears to be critical since a pure carbon
Figure 3 Forearm blood flow 6–8 h after exposure to filtered diesel or air. Infused (solid line) and non-infused (dashed line) forearm blood
flow in healthy subjects 6–8 h, following filtered diesel (grey circle) or air (open circle) exposure, during intra-brachial infusion of bradykinin,
acetylcholine, sodium nitroprusside, or verapamil: for all dose responses P , 0.0001. For filtered diesel exposure (grey circle) vs. air (open
circle); bradykinin, acetylcholine, sodium nitroprusside, and verapamil (P . 0.05 for all comparisons).
N.L. Mills et al.
Figure 4 Direct vascular effects of particles in rat aortic rings in vitro. Effect of diesel exhaust particles (filled circle), carbon nanoparticles (u)
(both 100 mg/mL), or vehicle (open circle) (Krebs buffer) on (A) phenylephrine contraction in mN, (B) phenylephrine contraction as a percen-
tage of maximum contraction (C) acetylcholine, and (D) sodium-nitroprusside-induced vasodilatation. Diesel exhaust particles did not affect
contraction to phenylephrine (P ¼ 0.64), but inhibited vasorelaxation to acetylcholine (***P , 0.001) and sodium nitroprusside (*P ¼ 0.019)
compared with control. Although there was an apparent rightward shift in the dose–response curve to acetylcholine and sodium nitroprusside
with carbon nanoparticles, vasorelaxation was not significantly affected (P ¼ 0.139 and P ¼ 0.224, respectively). Effect of (E) aqueous and (F)
organic separation of diesel exhaust particles on acetylcholine responses. (E) Aqueous-washed diesel exhaust particles (grey square), and
aqueous extract from diesel exhaust particles (open square), inhibit vasodilation to acetylcholine (**P , 0.01 for both), with the combined
effects of both constituents being equal to that of untreated particles (filled circle). (f) Organic-washed diesel exhaust particles (s) inhibited
acetylcholine induced vasodilatation (*P ¼ 0.010); the magnitude of this effect was not different from that of untreated diesel exhaust particles
(filled circle) (P ¼ 0.103). The organic extract of diesel exhaust particles (inverted triangle) had no effect on vasorelaxation to acetylcholine
(P ¼ 0.645). n ¼ 6 for all groups.
Nanoparticulate and vascular dysfunction
combustion-derived diesel exhaust nanoparticulate causes direct
vascular dysfunction in vitro and this appears to be attributable to
both soluble and insoluble fractions present on the surface of the
particulate. Taken together, our findings suggest that the adverse
vascular effects of diesel exhaust inhalation are predominantly
mediated by combustion-derived nanoparticulate. This provides a
rationale for testing environmental health interventions targeted at
reducing traffic-derived particulate emissions.
Several pollutants in diesel exhaust emissions are considered
harmful to human health including fine particles, nitrogen
dioxide, sulphur dioxide, and volatile organic compounds.1,21
While we have previously shown that exposure to nitrogen
dioxide, at levels in excess of those found in the present
study, does not alter vascular function,22it would not be possible
to assess the importance of all the components of diesel exhaust
individually and such a strategy would not account for potentially
important synergistic interactions between pollutants. Therefore,
our present study was designed specifically to determine the
role of nanoparticulate emissions, and whether the organic and
inorganic surface compounds, or the carbonaceous particles
themselves, are the main arbiter of the adverse cardiovascular
Figure 5 Reversal of the effects of particles in vitro by superoxide dismutase. (A) Diesel exhaust particles, (B) carbon nanoparticles, (C) Krebs-
washed diesel exhaust particles, (D) soluble extract from Krebs-washed diesel exhaust particles, and (E) dichloromethane-washed diesel
exhaust particles. Effect of (filled triangle) particles (100 mg/mL), (grey circle) superoxide dismutase (100 U/mL), (grey triangle) particles +
superoxide dismutase together, or vehicle (open circle) (Krebs buffer) on acetylcholine-induced vasodilatation. Superoxide dismutase reversed
the actions of all particles (*P , 0.05, **P , 0.01, ***P , 0.001) compared with particles alone, except nCB where the reversal by superoxide
dismutase was not significantly affected (P ¼ 0.18); n ¼ 6–9 for all groups. (f ) The hydroxyl radical scavenger mannitol had no effect on the
action of diesel exhaust particles on acetylcholine-induced relaxation (nsP ¼ 0.84: mannitol + diesel exhaust particles compared with diesel
exhaust particles alone). (filled triangle) Diesel exhaust particles (10 mg/mL), (diamond) mannitol (5 mM), (inverted triangle) diesel exhaust
particles + mannitol together, or vehicle (open circle) (Krebs buffer); n ¼ 6 for all groups.
N.L. Mills et al.
Exposure to dilute diesel exhaust for 2 h impaired vasomotor
vascular function with reduced vasodilatation in response to
both endothelium-dependent and -independent agonists. The
exposures are standardized to ensure a particle concentration of
300 mg/m3. These concentrations are found on a regular basis in
heavy traffic, occupational settings, and in the world’s most-
polluted cities. Exposure to 300 mg/m3for 1 h increases a
person’s average exposure during a 24-h period by only 12 mg/m3
and changes of this magnitude occur in even the least polluted
of cities on a daily basis.14Depletion of particles in diesel
exhaust using a highly efficient teflon filter reduced concentrations
by 1000-fold, and did not alter concentrations of the gaseous pol-
lutants of volatile organic species. We did not observe any impair-
ment of vascular function following exposure to filtered exhaust,
suggesting that the particles are essential in mediating the
adverse effects of diesel exhaust. While it is possible that the non-
particulate components of diesel exhaust have a synergistic role in
the presence of particulates, it is clear from our complementary in
vitro studies that diesel exhaust particles themselves have the
capacity to inhibit vascular function directly.
In the clinical studies, exposure to pure carbon nanoparticles did
not cause significant vascular dysfunction. In the pre-clinical exper-
iments, in vitro exposure to carbon nanoparticles caused a modest
inhibition of acetylcholine-induced relaxation, although this effect
was not statistically significant. Although carbon nanoparticles
cause pro-inflammatory effects in vitro,23the only previous study
to address the vascular effects of exposure to carbon nanoparticles
in man identified changes in circulating leucocyte expression of
adhesion molecules,24but no consistent effect on systemic vascu-
lar function.25These findings suggest that while exposure to ‘pure’
carbon nanoparticles can exert some systemic effects within the
cardiovascular system, their vascular actions are relatively small
in comparison with particles from vehicle exhaust. These obser-
vations are in keeping with our previous studies where exposure
to concentrated ambient particles, low in combustion-derived par-
ticulate, did not affect vascular function in either healthy subjects
or patients with coronary heart disease.26Taken together, these
studies suggest that particle composition is an important determi-
nant of the health effects of air pollution exposure.
The particles in diesel exhaust are laden with surface organic
compounds from unburned hydrocarbon fuels, and coated with
oxidized transition metals added to the fuels to improve effi-
Experimental studies have established that diesel
exhaust particles induce cellular oxidative stress and up-regulate
pro-inflammatory pathways.28Particle size, surface area, and
surface chemistry are thought to be important determinants of
these cellular effects. Whether inhaled nanoparticulate is capable
of translocation into the circulation in humans remains uncer-
tain,29,30but many of the chemical species on the surface of
these particles are hydrophilic, could diffuse across tight junctions
into the pulmonary interstitium, and be released into the circula-
tion to affect vascular function directly. In particular, the organic
species absorbed on the surface of combustion-derived particles
are chemically activeand potentially
Interestingly, removing the soluble and organic constituents
from the surface of diesel exhaust particles did not completely
abolish the effect on acetylcholine-mediated relaxation in vitro.
While dichloromethane removes aromatic components, such as
polyaromatic hydrocarbons, it is unclear to what extent neutral
non-polar organics (aliphatics) or the polar organics, such as qui-
nones, are effected by this treatment. It is possible that these com-
ponents are more important than aromatic hydrocarbons in
determining the adverse vascular effects of diesel exhaust particles.
At present, it is not possible to speculate further on the identity of
these harmful surface constituents, but a better understanding of
the detrimental components of diesel exhaust particulate will be
necessary for the future evaluation of technologies designed to
modify vehicle exhaust emissions.
Vasomotor dysfunction was associated with an increase in arter-
ial pressure that was not present in our previous studies.14,16In our
present study, subjects were exposed to each of the four con-
ditions for 2 h, where previously the exposures were limited to
1 h. It is possible that the longer duration of exposure results in
a more marked vascular pertubation with effects on systemic vas-
cular resistance and arterial pressure. Against this hypothesis, there
were no differences in basal vascular tone between exposures, and
systolic blood pressure was also raised following exposure to fil-
tered exhaust, perhaps suggesting that the vasomotor and
pressor effects are mediated by different mechanisms or com-
ponents of the exhaust. There is a substantial body of evidence
to supporta direct effect
pressure,33–36and the mechanisms that underpin this response
are beginning to emerge. Experimental studies suggest an impor-
tant role for vascular oxidative stress and the up-regulation of
The cellular pathways underlying the vascular impairment by
diesel particle exposure are important for understanding the
mechanism of action of particles and their expected impact on car-
diovacular system. The pattern of vascular dysfunction induced by
exposure to diesel exhaust was similar to that reported in our pre-
endothelium-dependent agonists and the nitric oxide donor
sodium nitroprusside. Together with the preserved response to
verapamil, this would suggest decreased bioavailability of nitric
oxide, or a decrease in the sensitivity of smooth muscle cells to
nitric oxide, mediates the effect of diesel particles. While decreases
in the activity of nitric oxide synthase appear to be at least partially
involved in the impaired vasodilator responses to endothelium-
dependent agonists,38,39vascular oxidative stress may also contrib-
ute to the reduced nitric oxide bioavailability.40Indeed, in vitro
studies provide support for this mechanism, where diesel
exhaust particles intrinsically generate free radicals, are able to
quench nitric oxide, and inhibit acetylcholine-mediated relaxation
of aortic ring preparations.18The involvement of superoxide free
radicals in the action of diesel particles (or their extracts) on vas-
cular function is further supported here by the ability of superox-
ide dismutase (but not the hydroxyl radical scavenger mannitol) to
reverse the vascular impairment. The incomplete reversal of the
actions of whole diesel exhaust particles may suggest that down-
stream pathways, such as alterations in calcium sensitivity or
up-regulation of rho-kinases,37may be partially involved in the
action of diesel exhaust particles. However, we note that pathways
downstream of soluble guanylate cyclase do not appear to be
of air pollutiononblood
in response to
Nanoparticulate and vascular dysfunction
affected by in vitro exposure to urban particulate in general.41
Further studies are required to more fully establish the underlying
mechanism for the vascular impairment and determine the effects
of exposure on the L-arginine-nitric oxide pathway in more detail.
Fibrinolytic function was not altered by any of the exposure
conditions. In previous work, we have demonstrated that exposure
to diesel exhaust generated from an idlling automobile engine
impaired endothelial t-PA release from the forearm of both
healthy subjects14and patients with coronary heart disease.16
We used a different exposure system in our current study, and
it is possible that differences between the exposures themselves
could account for this inconsistency. The electrical generator
used in the present study was running on commercially available
gas oil as opposed to the heavier diesel oil used previously. Com-
bustion was perhaps more efficient from the generator with a
greater proportion of diesel exhaust particles containing elemental
carbon rather than organics from unburnt fuel. Exposure con-
ditions were also associated with five-fold lower levels of both
carbon monoxide and nitrogen oxides than were present in our
previous studies.15,16,42The release of t-PA from granules in the
endothelium is not dependent on nitric oxide and down-regulation
of fibrinolytic function may be dependent on changes in protein
synthesis. We have previously found divergence in the effects of
exposure on vasomotor and fibrinolytic function at different time
points, with the effects on vasodilatation being more prominent
immediately after the exposure.
There are a number of potential limitations of this study that merit
consideration. We had intended to use a versatile aerosol concen-
tration enrichment system (VACES)26,43to expose subjects to
diesel exhaust particles in isolation. Unfortunately, it was not poss-
ible to reduce the concentration of gaseous co-pollutants suffi-
ciently to perform a useful comparison with unmodified dilute
diesel exhaust. We therefore elected to use a particle filtration
system to assess the effects of the gaseous co-pollutants in iso-
lation, and as such can only infer from our findings that diesel
exhaust particles are responsible for the adverse vascular effects
described in vivo.
To address this potential limitation, we undertook complemen-
tary in vitro studies. We acknowledge that the direct application of
particles to the vasculature in vitro makes a number of assumptions
regarding the feasibility of particle translocation, the numbers of
particles likely to translocate into the cardiovascular system and
changes to the nature of the particulate during this transit.18
Because of the vast number of potentially active constituents of
diesel exhaust particles, and the chemical processes needed to
fractionate these constituents, predictive in vitro assays are essential
to explore the mechanism of action (and interaction) of different
particle components. The concentrations of diesel exhaust particu-
late employed here are high, and it is unlikely that nanoparticles
would reach these concentrations in the systemic circulation
without a means of accumulation over a prolonged period.
However, the similarity of the vasodilator response in vitro to
that of the clinical studies is striking, both in the previous investi-
gations14,18and the present findings, suggesting that this method-
ology is representative of the vascular pathways activated in vivo.
We believe that this combined approach is justified and provides
additional insight into the components responsible for the
adverse vascular effects of diesel exhaust inhalation in man.
While we demonstrate that particle filtration abrogates the
effects of exposure to diesel exhaust, we used a highly efficient
teflon filter in our experimental system that would not readily
be applicable to the filtration of vehicle emissions or in air con-
ditioning systems. It would be important, therefore, to verify the
benefits of particle filtration using commercially available filtration
systems before these findings can guide environmental health
policy decisions on the use of retrofit particle traps.
Inhalation of dilute diesel exhaust (but not the associated gaseous
pollutants or carbon nanoparticles alone) impairs vascular function
in man. These findings suggest that the adverse vascular effects of
diesel exhaust are predominately mediated by combustion-derived
particles and provide a rationale for testing environmental health
interventionsto reduce particulate
whether they can reduce the incidence of cardiovascular events.
We thank Pamela Dawson, Daan Leseman, Finny Paterson and all
the staff at the Wellcome Trust Clinical Research Facility, Edin-
burgh, for their assistance with the studies. We also acknowledge
the support of the British Heart Foundation Centre of Research
Excellence (CoRE) award and UK National Health Service
(NHS) Research Scotland (NRS), through NHS Lothian and the
Chief Scientist Office.
This work was supported by a British Heart Foundation Programme
Grant [PG/10/009]. NLM was supported by an Intermediate Clinical
Research Fellowship from the British Heart Foundation [FS/10/024]
and Programme Grant (PG/05/003), UK. Dutch Ministry of Housing,
Spatial Planning and Environment (VROM), the Netherlands. Funding
to pay the Open Access publication charges for this article was pro-
vided by the British Heart Foundation.
Conflict of interest: No conflicts of interest to disclose. The study
complies with the Declaration of Helsinki and has been approved by
all relevant local (UK) Ethics Committees.
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Nanoparticulate and vascular dysfunction