Published ahead of print on April 19, 2007, doi:10.1164/rccm.200606-872OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200606-872OC
Persistent Endothelial Dysfunction in Humans after Diesel Exhaust Inhalation1 Department of Respiratory Medicine and Allergy, Umeå University, Umeå, Sweden; 2 Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom; 3 Department of Medicine, Umeå University, Umeå, Sweden; 4 Free Radical Research Facility, UHI Millennium Institute, Inverness, United Kingdom; and 5 ELEGI Colt Laboratory, Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom Correspondence and requests for reprints should be addressed to Anders Blomberg, M.D., Ph.D., Department of Respiratory Medicine and Allergy, Umeå University Hospital, SE-901 85 Umeå, Sweden. E-mail: anders.blomberg{at}lung.umu.se
Rationale: Exposure to combustion-derived air pollution is associated with an early (1–2 h) and sustained (24 h) rise in cardiovascular morbidity and mortality. We have previously demonstrated that inhalation of diesel exhaust causes an immediate (within 2 h) impairment of vascular and endothelial function in humans. Objectives: To investigate the vascular and systemic effects of diesel exhaust in humans 24 hours after inhalation. Methods: Fifteen healthy men were exposed to diesel exhaust (particulate concentration, 300 µg/m3) or filtered air for 1 hour in a double-blind, randomized, crossover study. Twenty-four hours after exposure, bilateral forearm blood flow, and inflammatory and fibrinolytic markers were measured before and during unilateral intrabrachial bradykinin (100–1,000 pmol/min), acetylcholine (5–20 µg/min), sodium nitroprusside (2–8 µg/min), and verapamil (10–100 µg/min) infusions.
Measurements and Main Results: Resting forearm blood flow, blood pressure, and basal fibrinolytic markers were similar 24 hours after either exposure. Diesel exhaust increased plasma cytokine concentrations (tumor necrosis factor- Conclusions: Twenty-four hours after diesel exposure, there is a selective and persistent impairment of endothelium-dependent vasodilatation that occurs in the presence of mild systemic inflammation. These findings suggest that combustion-derived air pollution may have important systemic and adverse vascular effects for at least 24 hours after exposure.
Key Words: air pollution • endothelium • blood flow • inflammation • diesel exhaust
The link between ambient particulate matter (PM), air pollution, and increased cardiorespiratory mortality and morbidity is well established (1). Short-term exposure to traffic and ambient air pollution is associated with an increased risk of early (1–2 h) and delayed (24 h) presentation with acute myocardial infarction (2, 3), or rehospitalization for myocardial ischemia in patients with prior myocardial infarction (4). Long-term repeated exposure to PM pollution increases the risk of cardiovascular mortality, with deaths attributable to ischemic heart disease, arrhythmia, heart failure, and cardiac arrest (5–7). These associations are strongest for fine particulate air pollutants (  2.5 µm in diameter; PM2.5) (8). Diesel exhaust emissions are a significant source of PM2.5 in urban environments, particularly in Europe where the use of diesel engines in transport has increased steadily in recent years (9). As a consequence, diesel exhaust exposures have been used as models of PM pollution in experimental studies (10–12). The biological mechanisms underlying the cardiovascular effects of PM air pollution are largely unknown, although it has been suggested that pulmonary inflammation results in systemic consequences that adversely affect the cardiovascular system (13). In vitro studies, animal models, and human exposures have clearly established the oxidant and proinflammatory nature of combustion-derived PM and implied a role for oxidative stress in determining the toxicity of ambient air pollution and the proinflammatory effects of diesel exhaust particulates (14–16). At levels encountered in an urban environment, we have previously demonstrated that exposure to diesel exhaust causes pronounced airway inflammation, including recruitment of inflammatory cells, the up-regulation of vascular endothelial adhesion molecules, and the enhanced epithelial expression of cytokines. These effects are associated with the up-regulation of important oxidative stress-related transcription factors and mitogen-activated protein (MAP) kinases in the bronchial epithelium (11, 12, 17–20). Endothelial dysfunction is widely considered to represent the earliest pathologic process in atherosclerosis (21), with established risk factors for cardiovascular disease adversely affecting endothelial function (22, 23). In recent studies, we have demonstrated an immediate impairment of vascular and endogenous fibrinolytic function in young healthy volunteers after exposure to diesel exhaust (24). In the absence of systemic inflammation up to 6 hours after exposure, we suggested that these early vascular effects were determined by oxidative stress. The duration of these adverse vascular effects are unknown, and the potential for developing later pulmonary and systemic inflammatory effects to potentiate vascular dysfunction requires further investigation. The aim of the present study was to investigate whether there is systemic inflammation and sustained vascular dysfunction in healthy volunteers 24 hours after exposure to diesel exhaust.
Study Design Fifteen healthy male nonsmokers (mean age, 26 yr; range, 18–38 yr) participated in the study. Subjects were exposed to filtered ambient air or diesel exhaust at a particulate concentration of 300 µg/m3, in a randomized, double-blind, crossover fashion for 1 hour, according to a previously described standard protocol (11). During each exposure, subjects alternated moderate exercise on a bicycle ergometer and rest at 15-minute intervals. Vascular assessments were performed 24 hours after each exposure, with at least 2 weeks allowed between exposures. Subjects also underwent an identical vascular assessment 2 to 4 hours after each exposure; the results of this assessment are reported in full elsewhere (24). The study was performed in accordance with the Declaration of Helsinki, with the approval of the local ethics committee, and with the written, informed consent of each volunteer.
Diesel Exposure
Vascular Study Venous cannulae (17-gauge) were inserted into both arms. Blood was drawn simultaneously from each arm at baseline and during the infusion of each dose of bradykinin, and collected for estimation of plasma t-PA and plasminogen activator inhibitor type 1 (PAI-1) concentrations. Hematocrit was determined at baseline and after infusion of bradykinin at 1,000 pmol/minute.
Systemic Inflammation and Oxidative Stress
Data Analysis and Statistics
There were no differences in resting heart rate, blood pressure, or baseline forearm blood flow between or during the two study visits (Table 1).
Vascular Function After both air and diesel exhaust exposures, there were dose-dependent increases in the infused forearm blood flow with all vasodilators (p < 0.001 for all). After exposure to diesel exhaust, endothelium-dependent vasodilatation was reduced with acetylcholine (p = 0.01) and appeared to be reduced with bradykinin (p = 0.08; Figure 1). In contrast, there were no effects on endothelium-independent vasodilatation: peak blood flow responses to sodium nitroprusside (14.2 ± 1.2 vs. 12.8 ± 0.8 ml/100 ml/min) and verapamil (14.6 ± 0.9 vs. 13.4 ± 0.9 ml/100 ml/min) were similar.
Venous plasma concentrations of nitrite were not effected by exposure (Table 2).
Bradykinin caused a dose-dependent increase in plasma t-PA concentrations (p < 0.001; Table 3), which was unaffected by diesel exhaust exposure. The estimated net t-PA antigen release was similar after exposure to both diesel exhaust and filtered air (Figure 2).
All subjects also underwent an identical early vascular assessment at 2 to 4 hours after exposure, the results of which are published in full elsewhere (24). Peak forearm blood flow response to endothelium-dependent vasodilators correlates well between the early (2–4 h) and late (24–26 h) vascular assessments for bradykinin (r = 0.60, p < 0.05) and acetylcholine (r = 0.72, p < 0.05). There was also a relationship between the magnitude of impairment caused by exposure to diesel exhaust (difference between peak forearm blood flow after diesel exhaust and filtered air) at the early and late time points for bradykinin (r = 0.58, p < 0.05), but not for the other vasodilators.
Systemic Inflammation and Oxidative Stress
The majority of inflammatory markers were significantly reduced at 24 hours when compared with baseline after exposure to both filtered air and diesel exhaust. This consistent response across exposures is due to an effect of one or more of the study interventions, which included a period of aerobic exercise, a regulated healthy diet, a prolonged fast, and repeated blood sampling. Significant differences between exposures occurred with absolute measures at 24 hours and change from baseline in IL-6, TNF- Suspensions of diesel exhaust particles showed a time-dependent increase in the characteristic three-peak EPR spectrum for a spin-adduct with the unpaired electron in the vicinity of a nitrogen atom (i.e., 4-oxo-tempo, the oxidized form of Tempone-H) (Figure 3a). The signal increased at a constant rate over the 60-minute period. EPR measurements were approximately fivefold higher in suspensions that contained diesel filters (blank, 1,144 units; diesel, 5,864 units; t = 60 min; Figure 3b). Superoxide dismutase (SOD) inhibited the EPR signal from diesel, causing a 30.2% reduction in signal. This concentration of SOD had a similar magnitude of effect on the diesel signal as it did to that of the superoxide generator pyrogallol (34.7% reduction; Figure 3b).
Twenty-four hours after a transient 1-h exposure to diesel exhaust, there is a selective and persistent impairment of endothelium-dependent vasodilatation that occurs in the presence of mild systemic inflammation after a period of potential oxidative stress. These findings suggest that combustion-derived air pollution may have important adverse systemic and vascular effects for at least 24 hours after exposure. This may account for the epidemiologic observations of recent time-series studies suggesting both an acute (1–2 h) and a sustained peak in adverse cardiovascular outcomes after exposure to air pollution. Exposure to air pollution causes airway inflammation and has an important negative effect on respiratory health. Diesel exhaust causes neutrophilic airway inflammation 6 to 24 hours after exposure (11, 12, 27), increases airway antioxidant defenses, and activates redox-sensitive transcription factors in vivo, consistent with oxidative stress–induced and cytokine-mediated inflammation (20, 28). It is increasingly recognized that these effects may induce important systemic effects (12, 29), including vascular inflammation (30). We hypothesized that our initial observations of an immediate (within 2 h) impairment of vascular function were due to the oxidative effects of diesel exhaust (24). After exposure to diesel exhaust, endothelium-dependent and endothelium-independent nitric oxide donors caused reduced vasodilatation, whereas the endothelium- and nitric oxide–independent vasodilator verapamil caused normal vasodilatation. This pattern of vascular dysfunction suggested increased consumption of nitric oxide, whether it be endogenously derived from endothelial nitric oxide synthase or from an exogenous source, such as sodium nitroprusside. In the current study, we demonstrate a persistent endothelium-dependent vascular dysfunction 24 hours after an hour-long exposure to diesel exhaust. Although vasodilatation to both endothelium-dependent agonists appeared to be impaired, this only reached statistical significance for acetylcholine. Bradykinin causes vasodilatation through the release of various endothelium-derived factors, including nitric oxide, although it is believed that hyperpolarizing factor is the primary mediator of this response in humans (31). It is possible that differences in acetylcholine- and bradykinin-mediated vasodilatation may be explained by variation in the relative contribution of nitric oxide to the vasomotor response of these agonists. The mechanism of this selective impairment of endothelium-dependent vasodilatation has not been determined, but we suggest that this may be due to modification of endothelial homeostatic pathways after an initial oxidative burst. The role of vascular oxidative stress in mediating endothelial dysfunction in this clinical model requires confirmation. However, the mechanism is supported by in vitro studies (32–34), as well as human exposure studies by our own group (25) and others (35). The endothelium is a major target of oxidative stress and this interaction plays an important role in the pathophysiology of vascular disease (36). Incubation of aortic ring preparations with diesel exhaust particles inhibits acetylcholine-mediated relaxation, an effect that can be reversed by coincubation with the free radical scavenger SOD (32). Furthermore, diesel exhaust particles can induce oxidative modification of low-density lipoprotein, the major determinant of atheromatous vascular disease (37). Diesel exhaust is a complex mixture of gases and particles, and from our findings we cannot exclude a role for nonparticulate or soluble components. The most abundant gaseous pollutants produced in the combustion of diesel fuel are oxides of nitrogen. In epidemiologic studies, ambient nitrogen dioxide (NO2) is considered a surrogate for traffic-derived pollution, with PM held responsible for the majority of the adverse health effects of air pollution (38). Although the direct effect of NO2 on vascular function has not been studied to date, exposure to NO2 alone at a higher concentration and for a longer duration than used here does not induce an inflammatory response in the airway mucosa (39). In contrast, exposure to dilute diesel exhaust for 1 hour at similar particulate concentrations to the present study resulted in pronounced airway mucosal inflammation with up-regulation of neutrophils, mast cells, and T lymphocytes (11). Although this suggests that the particulate phase of dilute diesel exhaust is responsible for the adverse inflammatory (11) and vascular effects of diesel exhaust (24), further proof is required with additional control exposures to nitrogen oxides and filtered diesel exhaust. Recent findings by Campen and colleagues, in which incubation with soluble components of diesel exhaust caused vascular dysfunction in isolated coronary artery rings from mice, highlight the need for further controlled exposure studies (40). Whether diesel particulates or soluble components of the exposure, including organic hydrocarbons and transition metals, can directly affect the systemic vascular endothelium after inhalation also requires clarification. Although evidence that inhaled nanoparticles can translocate into the circulation in humans remains controversial (41, 42), it is not in doubt that diesel exhaust particulates are capable of inducing oxidative stress in vitro, with reactive oxidant species arising from the redox potential of the particles themselves and from the activation of inflammatory cells. Using EPR, we demonstrate that diesel exhaust particulate is capable of generating oxidative free radicals without prior interaction with pulmonary or vascular tissue. Furthermore, coincubation of diesel particles with SOD partially prevented this response, indicating a contribution of superoxide to this oxidative signal. However, measuring systemic oxidative stress in vivo is difficult, because the oxidative state is modulated by a range of antioxidant defenses (17). Interestingly, we demonstrate an increase in the antioxidant capacity of plasma 24 hours after exposure to diesel exhaust, perhaps suggesting up-regulation of antioxidant defense mechanisms after earlier systemic oxidative stress. In contrast to our previous study, stimulated release of endothelial t-PA from the forearm circulation was not impaired at 24 hours (24). In health, the vascular endothelium delicately balances regulatory pathways controlling coagulation, fibrinolysis, and inflammation, as well as regulating vascular tone. It is perhaps not surprising that these complex dynamic functions are altered by exposure to diesel exhaust at different time points. Endogenous fibrinolytic function was impaired at 6 to 8 hours in our previous studies, but normalized at 24 hours, suggesting that this aspect of endothelial homeostasis recovers earlier than vasomotor function after exposure to air pollution.
We did not find evidence of a systemic cellular inflammatory response, but we did identify changes in proinflammatory cytokines IL-6 and TNF- Endothelial dysfunction, characterized as an impaired vasodilatation to acetylcholine, predicts the likelihood of future cardiovascular events and death in patients with coronary artery disease (48) and in at-risk individuals with normal coronary arteries (49). Although the mechanism of this association has not been precisely identified, this vascular dysfunction clearly has important clinical implications. Our findings of endothelial dysfunction 24 hours after diesel exhaust inhalation suggest that the adverse cardiovascular effects of combustion-derived air pollution are mediated through persistent detrimental vascular effects.
Conclusions
The authors thank Pamela Dawson, Chris Ludlam, Frida Holmström, Annika Johansson, Maj-Cari Ledin, Jamshid Pourazar, Ann-Britt Lundström, Margot Johansson, Mona Svensson, Ester Roos-Engstrand, and all the staff in the Department of Respiratory Medicine and Allergy and Clinical Research Facility, University Hospital, Umeå, and Wellcome Trust Clinical Research Facility, Edinburgh, for their assistance with the studies.
Supported by the Swedish Heart-Lung Foundation; the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS); Front Research, the County Council of Västerbotten, Sweden; Umeå University, Sweden; British Heart Foundation program grant RG/05/003; and the British Cardiac Society (Michael Davies Research Fellowship to N.L.M.).
* These authors contributed equally to this article. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200606-872OC on April 19, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form June 29, 2006; accepted in final form April 19, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
and interleukin-6, p < 0.05 for both) but appeared to reduce acetylcholine (p = 0.01), and bradykinin (p = 0.08) induced forearm vasodilatation. In contrast, there were no differences in either endothelium-independent (sodium nitroprusside and verapamil) vasodilatation or bradykinin-induced acute plasma tissue plasminogen activator release. 
