Particulate Matter Air Pollution and Cardiovascular Disease: An Update to the Scientific Statement From the American Heart Association

Time-series and case-crossover studies explore associations between short-term changes in air pollution and daily changes in death counts. The sum of current evidence supports the findings of an earlier review²⁸ that demonstrated that short-term elevations in daily PM levels lead to a greater absolute risk for CVD-related mortality than for all other causes. Even if similar acute RR elevations (≈1.01) are estimated between cardiovascular and pulmonary mortality, CVDs account for 69% of the increase in absolute mortality rates compared with 28% for pulmonary diseases attributable to short-term PM exposure. Recently, more rigorous modeling techniques have been used in attempts to better estimate pollution-mortality associations while controlling for other time-dependent confounding covariables.^29,30 There have been well over 100 published daily time-series studies reporting small but statistically significant PM-mortality associations that have been the subject of quantitative reviews or meta-analyses.^3,27,31–33 Table 2 summarizes recent multicity analyses and studies published since 2004.

To address concerns about city selection bias, publication bias, and influences of copollutants, several large, multicity, daily time-series studies have been conducted worldwide. One of the largest was the National Morbidity, Mortality, and Air Pollution Study (NMMAPS). Published reports from this study included as few as 20 US cities,^44,45 as many as 100 cities,^46,47 and more recently, data for hundreds of counties (Table 2).⁴⁸ The observed relationship between PM exposure and excess mortality remained independent of several gaseous copollutants (NO₂, CO, or SO₂). Recent analyses suggest that O₃ may also independently contribute to cardiopulmonary mortality risk^49,50; however, coexposures to secondary particle pollutants may be responsible in part for this latter association.⁵¹

Several studies have also been conducted outside the United States, including the Air Pollution and Health: A European Approach (APHEA and APHEA-2) projects, which examined daily PM-related mortality effects in multiple cities.^36,52 PM air pollution was significantly associated with daily mortality counts for all-cause, cardiovascular, and respiratory mortality (Table 2). Further analyses of the European data suggest that CVD deaths are also associated with exposure to NO₂⁵³ and CO.⁵⁴ A few new time-series studies have also confirmed similar increases in cardiovascular mortality related to short-term PM exposure in China^55–57 and Bangkok, Thailand.⁴² Additional multicity studies have been conducted worldwide with analyses of CVD deaths (Table 2).^{38–42,58–60} Finally, in a recent analysis that included several Asian cities, SO₂, NO₂, O₃, and PM₁₀ were all associated with excess cardiovascular mortality.⁴²

In an attempt to evaluate the coherence of multicity studies across continents, the Air Pollution and Health: A Combined European and North American Approach (APHENA) study analyzed data from the APHEA, NMMAPS, and Canadian studies.⁶¹ The combined effect on all-cause mortality ranged from 0.2% to 0.6% for a 10-μg/m³ elevation in daily ambient PM₁₀, with the largest effects observed in Canada. Among individuals older than 75 years, the effects were greater for cardiovascular mortality than for overall and pulmonary mortality (0.47% to 1.30%). Older age (>75 years) and higher rates of unemployment were related to greater PM mortality risks in both continents. Higher NO₂ levels were associated with larger PM₁₀ effects on mortality, particularly in Europe. Finally, there appeared to be no lower-limit threshold below which PM₁₀ was not associated with excess mortality across all regions.

Although short-term changes in PM concentrations have deleterious health effects, longer-term exposures may have a more pertinent clinical health effect on cardiovascular morbidity and mortality given that individuals are typically exposed to higher air pollution levels over extended periods of time. An additional source of exposure variability that has been exploited in epidemiological studies is spatial variability, which includes differences in average ambient concentrations over extended periods of time across metropolitan areas or across smaller communities within local areas. Recent emphasis has been on prospective cohort studies that control for individual differences in multiple confounding variables and cardiovascular risk factors. A summary of these studies is presented in Table 3 and Figure 1. These cohort studies generally demonstrate larger overall mortality effects than the results of time-series analyses.

Two landmark cohort-based mortality studies, the Harvard Six Cities⁶² and the ACS studies,⁶⁶ were reported in the mid 1990s and were discussed previously.¹ In both, PM_2.5 and sulfate particulate pollution were associated with increases in all-cause and cardiopulmonary disease (Table 3). In addition, intensive independent reanalyses⁶³ corroborated the original findings of both studies and resulted in innovative methodological contributions that demonstrated the robustness of the results to alternative modeling approaches. In both the Harvard Six Cities^62,64 and the ACS⁶⁷ studies, PM air pollution–related mortality was substantially higher for cardiovascular- than for pulmonary-related causes.

Since 2004, there have been further analyses of both studies. Laden et al⁶⁴ extended the mortality follow-up of the Harvard Six Cities cohort for an additional 8 years. PM_2.5 associations, similar to those found in the original analysis, were observed for all-cause and CVD mortality (Table 3). Furthermore, reductions in PM_2.5 concentrations for the extended follow-up period were associated with reduced mortality risk. Further analysis suggested that the health effects of changes in exposure were seen primarily within 2 years.⁸⁴ In addition to confirming the earlier mortality relationship, the recent observations suggest that the adverse health effects mediated by longer-term PM air pollution exposure can be estimated reasonably accurately by the previous few years of particle levels.

Extended analyses of the ACS cohort that emphasize efforts to control for the effects of other covariates and risk factors have corroborated the previously reported mortality associations with particulate and sulfur oxide pollution.⁶⁸ Elevated mortality risks were most strongly associated with PM_2.5. Coarse particles (PM_10–2.5) and gaseous pollutants, except for SO₂, were generally not significantly related to mortality. In another extended analysis,⁶⁷ the death certificate classifications of underlying causes of death due to PM_2.5 exposures were observed to be principally ischemic heart disease, arrhythmias, heart failure, and cardiac arrest. Finally, recent additional analyses attempted to control for the fact that variations in exposure to air pollution across cities or within cities may correlate with socioeconomic or demographic gradients that influence health and susceptibility to environmental exposures.^85,86 When controlled for individual risk factor data, the mortality associations for intrametropolitan PM_2.5 concentration differences within the Los Angeles, Calif, area were generally larger than those observed in the full cohort across metropolitan areas.⁶⁹ However, the results were somewhat sensitive to the inclusion of zip code–level ecological variables, which suggests potential contextual neighborhood confounding. Krewski et al⁷⁰ subsequently observed that full adjustments for multiple ecological covariates did not reduce the estimated PM_2.5-related mortality effect. The association for ischemic heart disease mortality in particular was highly robust across various study areas and modeling strategies and after controlling for both individual and ecological covariates.

An additional recent analysis of the ACS cohort evaluated the health effects of ozone compared with PM_2.5.⁸⁷ The findings reconfirmed the independent cardiovascular mortality increase related to fine-particle exposure. However, after adjustment for PM_2.5, ozone was associated solely with an elevated risk of death due to respiratory causes; there was no independent risk of ozone exposure on CVD-related mortality. This suggests that the positive findings reported in NMMAPS⁵⁰ regarding cardiopulmonary mortality and short-term ozone exposure could be explained at least in part by the enhanced risk of mortality due to lung disease categories.

Several additional cohort studies have been published in the past few years (Table 3). Eftim and colleagues⁶⁵ studied 2 very large “cohorts” of US Medicare participants who lived in locations included in the Harvard Six Cities and ACS studies. Effects of PM_2.5 exposure on mortality for the period 2000 to 2002 were estimated after controlling for multiple factors, although not at the individual patient level. For all-cause mortality, the PM_2.5-mortality associations were larger than those observed in the Harvard Six Cities or ACS cohorts. In an additional analysis of 13.2 million US Medicare participants for the time period 2000 to 2005,⁷¹ PM_2.5-mortality associations were shown to be similar to those observed in the Harvard Six Cities and ACS studies in the East and Central regions of the United States (and when the data were pooled for the entire United States). However, PM_2.5 was not associated with mortality in the Western United States or for the oldest age group (>85 years old). These findings generally corroborate the earlier cohort studies and add evidence that aspects of exposure (PM sources or composition) and patient susceptibility might play important roles in determining the health risks.

In a cohort of postmenopausal women without prior CVD from the Women’s Health Initiative Observational Study,⁷² an association between longer-term PM_2.5 exposure (median follow-up of 6 years) and cardiovascular events (primary end point) was observed. After adjustment for age and other risk factors, an incremental difference of 10 μg/m³ PM_2.5 was associated with a 24% (95% confidence interval [CI]9% to 41%) increase in all first cardiovascular events (fatal and nonfatal, with a total of 1816 cases). Notably, an incremental difference of 10 μg/m³ PM_2.5 was also associated with a large 76% (95% CI 25% to 147%) increase in fatal cardiovascular events, based on 261 deaths. The risks for both coronary heart disease and strokes were found to be similarly elevated. Interestingly, within-city PM_2.5 gradients appeared to have larger cardiovascular effects than those between cities, although this difference was not statistically significant. Finally, overweight women (body mass index >24.8 kg/m²) were at relatively greater cardiovascular risk due to particulate air pollution than leaner women. Noteworthy aspects of this study were improved assessment of the end points by medical record review (rather than by death certificate) and long-term particle exposure estimation. The control for individual-level confounding variables was also superior to that of previous cohort studies.

In another cohort of women, a subset of the Nurses’ Health Study from the northeastern United States,⁷³ an increase of 10 μg/m³ modeled estimates of PM₁₀ exposures was associated with an approximately 7% to 16% increased risk of all-cause mortality and a 30% to 40% increase in fatal coronary heart disease, depending on the level of adjustment for covariates. This study found that the strongest health risks for all-cause and cardiovascular mortality were seen in association with the average PM₁₀ exposure during the previous 24 months before death. Similar to the findings of the Women’s Health Initiative, the cardiovascular mortality risk estimates were larger than those of previous cohort studies. In addition, obese women (body mass index >30 kg/m²) were at greater relative risk, and the increases in mortality (all-cause and cardiovascular) were larger than the effects on nonfatal events. The results were also in accordance with the latest Harvard Six Cities analyses⁶⁴ that show that exposure over the most recent preceding 1 to 2 years can accurately estimate the majority of the health risks due to longer-term PM air pollution exposures.

The pollution-mortality association has also been assessed in several other cohort studies in the United States and Europe (Table 3).^76–83 In a recent analysis of the Adventist Health Study of Smog (AHSMOG) cohort with a much longer follow-up than the original studies,^74,88 fatal coronary heart disease was significantly associated with PM_2.5 among females but not males.⁷⁵ These observations along with the remarkably robust health effects in the Women’s Health Initiative Observational Study and Nurses’ Health Study suggest that women may be at special risk from PM exposure. The overall cohort study evidence demonstrates that a 10-μg/m³ increase in PM_2.5 exposure is in general positively associated with excess mortality, largely driven by increases in cardiopulmonary or cardiovascular deaths (Figure 1). Independent results from the Women’s Health Initiative Study,⁷² the US Medicare cohorts,⁷¹ the German women cohort,⁸⁰ and the intracity Oslo (Norway) study⁸¹ contribute substantially to this evidence. Although the Dutch cohort,⁸² AHSMOG,^74,75 French PAARC (Pollution Atmosphérique et Affections Respiratoires Chroniques [air pollution and chronic respiratory diseases]),⁷⁹ Veterans Affairs hypertensive male study,⁷⁷ and 11 CA county⁷⁸ studies observed increased mortality risks associated with higher PM_2.5 exposure that were statistically significant in some analyses, the observed health risks were less robust. A finding that is somewhat consistent across the Veterans Affairs hypertensive male study,⁷⁷ 11 CA county,⁷⁸ Oslo,⁸¹ and US Medicare cohorts⁷¹ is that the PM_2.5-mortality effect estimates tend to decline with longer periods of follow up or in a substantially older cohort. These studies also often observed elevated mortality risks according to alternative indicators of air pollution exposure, especially metrics of traffic-related exposure.

Excess cardiovascular mortality and increased rates of hospitalizations are similarly associated with day-to-day changes in PM air pollution (Tables 2 and 4). However, significant differences between geographic regions in the risk relationships have been observed, and more investigation is required to explain this heterogeneity.

Among the cohort studies that provided relevant results, the ACS study found a relationship between increased risk for ischemic heart disease death and long-term exposure to elevated PM_2.5 levels (Table 3).^67,69,106 Indeed, ischemic cardiac events accounted for the largest relative (RR 1.18, 95% CI 1.14 to 1.23) and absolute risk for mortality per 10-μg/m³ elevation in PM_2.5.⁶⁷ A survival analysis of US Medicare data for 196 000 survivors of acute MI in 21 cities showed the risk of an adverse post-MI outcome (death, subsequent MI, or first admission for congestive heart failure) was increased with higher exposure to PM₁₀.¹⁰⁷ Data from the Worcester Heart Attack study also found that long-term exposure to traffic-related air pollution was associated with significantly increased risk of acute MI.¹⁰⁸ However, in the Women’s Health Initiative⁷² and the Nurses’ Health Study,⁷³ only disease categories that included fatal coronary events, but not nonfatal MI alone, were statistically elevated in relation to PM_2.5. The effect size for cardiovascular mortality was much larger and much more statistically robust than for nonfatal events such as MI in both studies.

Various time-series and case-crossover studies have also reported increased ischemic heart disease hospital admissions associated with short-term elevated concentrations of inhalable and/or fine PM air pollution.^31,40,103 In the US Medicare study, a reduction of PM_2.5 by 10 μg/m³ was estimated to reduce ischemic heart disease admissions in 204 counties by 1523 (95% posterior interval 69 to 2976) cases per year.¹⁰³ Several studies have also found positive associations between elevated PM or traffic exposures over a period as brief as a few hours^109–111 or a few days and an elevated risk for MI (Table 5).^{13,110,112–115} In general, acute increases in risk for ischemic heart disease events have been observed consistently, even as rapidly as 1 to 2 hours after exposure to elevated PM, in case-crossover analyses.^109–111 Other studies have reported an increased risk for MI shortly after exposure to traffic. Peters et al¹¹¹ reported in 691 subjects in Augsburg, Germany, a strong association (odds ratio 2.92, 95% CI 2.22 to 3.83) between onset of MI and traffic exposure within the past hour, although whether this was a result of the air pollution or a combination of other factors (eg, noise and stress) is not certain. Additional analyses did not report an association between recent UFP exposures and MI onset; however, the levels of PM_2.5 and several gaseous pollutants 2 days earlier were related to MI risk.¹¹⁶ The lack of relationship between MI and UFPs may be due to the fact that the levels were measured regionally and remote from the localized source and may therefore reflect exposure misclassification. Finally, in the only study in which participating subjects had coronary angiograms performed previously, ischemic cardiac events were found to occur in relation to PM air pollution exposure solely among individuals with obstructive coronary atherosclerosis in at least 1 vessel.¹³ This finding suggests the importance of patient susceptibility (eg, the presence of preexisting coronary artery disease) for PM to trigger an acute ischemic event within hours to days after exposure.

In the ACS cohort study, it appeared that deaths due to arrhythmias, heart failure, and cardiac arrest (RR 1.13, 95% CI 1.05 to 1.21 per 10 μg/m³) were also associated with prolonged exposure to PM_2.5, although not as strongly as ischemic heart disease mortality,⁶⁷ although potential mortality misclassification on death certificates makes the actual cause of death not entirely certain in all circumstances. Heart failure rates or mortality associations were not reported in the other cohort studies.

Daily hospitalizations for heart failure have also been associated with short-term changes in PM exposure.³¹ Heart failure associations with PM were observed in a large daily time-series analysis of PM_2.5 and cardiovascular and respiratory hospitalizations by use of a national database constructed from US Medicare files.¹⁰³ A 10-μg/m³ increase in concurrent-day PM_2.5 was associated with a 1.28% (95% CI 0.78% to 1.78%) increase in heart failure admissions, the single largest cause for hospitalization in this cohort. A reduction of PM_2.5 by 10 μg/m³ was estimated to reduce heart failure admissions in 204 counties by 3156 (95% posterior interval 1923 to 4389) cases per year.¹⁰³ Another analysis in Medicare recipients in 7 US cities found a 10-μg/m³ increase in concurrent-day PM₁₀ was associated with a 0.72% (95% CI 0.35% to 1.10%) increase in heart failure admissions.¹¹⁷ Traffic-related air pollution has also been shown to be significantly associated with increased mortality risk after acute heart failure.¹¹⁸ Finally, a study from Utah’s Wasatch Front area explored longer lagged-exposure periods and found that a 14-day lagged cumulative moving average of 10 μg/m³ PM_2.5 was associated with a 13.1% (95% CI 1.3% to 26.2%) increase in heart failure admissions.¹¹⁹

Among the cohort studies that provided pertinent results, the Women’s Health Initiative reported significant increases in both nonfatal stroke (hazard ratio 1.28, 95% CI 1.02 to 1.61) and fatal cerebrovascular disease (hazard ratio 1.83, 95% CI 1.11 to 3.00) per 10-μg/m³ elevation in prolonged exposure to PM_2.5.⁷² However, no significant association between stroke mortality and PM air pollution was found in the ACS study.⁶⁷

Several studies have also reported small but statistically significant associations between short-term PM exposure and cerebrovascular disease. Daily time-series studies of stroke mortality in Seoul, Korea,^120,121 observed that elevated air pollution (including measures of PM, NO₂, CO, and O₃) was associated with increases in stroke mortality. When analyzed separately by stroke type,¹²¹ the pollution association was associated with ischemic but not hemorrhagic stroke. Risk of stroke mortality was also associated with daily increases in PM₁₀ and NO₂ in Shanghai, China.⁵⁶ A daily time-series study in Helsinki, Finland,¹²² found that PM_2.5 and CO were associated with stroke mortality in the warm but not the cold seasons. Several studies have also observed increased stroke or cerebrovascular hospital admissions associated with increased exposure to PM or related pollutants.^{31,38,40,46,123–125} For example, a study of hospital admissions for Medicare recipients in 9 US cities¹²⁵ found that several measures of air pollution (PM₁₀, CO, NO₂, and SO₂) 0 to 2 days before admission were associated with ischemic but not hemorrhagic stroke. Studies of ischemic stroke and transient ischemic attacks based on population-based surveillance have also been conducted in Dijon, France,¹²⁶ where O₃ exposure (but not PM₁₀) was associated with ischemic stroke, and in Corpus Christi, Tex,¹²⁷ where both PM_2.5 and O₃ were associated with ischemic strokes and transient ischemic attacks.

There have been only a few studies that have explored a relationship between air pollution and peripheral vascular diseases. Studies using Medicare data for 204 US counties observed nearly statistically significant positive associations between daily changes in measures of PM pollution and hospitalizations for peripheral vascular diseases.^103,104 The ACS cohort found no association between other atherosclerotic and aortic aneurysm deaths and long-term PM_2.5 exposure.⁶⁷

Recently, a case-control study from the Lombardy region of Italy found a 70% increase in risk of deep vein thrombosis per 10-μg/m³ elevation in long-term PM₁₀ level.¹²⁸ This is the first observation that particulate air pollution can enhance coagulation and thrombosis risk in a manner that adversely affects the venous circulation in addition to the arterial cardiovascular system.

Several studies have observed associations between fine PM and related pollutants and cardiac arrhythmias, often based on data from implanted cardioverter-defibrillators.^129–136 However, no clear pollution-related associations were observed in studies from a relatively clean metropolitan area, Vancouver, British Columbia, Canada,^137,138 or from a relatively large study in Atlanta, Ga.¹³⁹ Similarly, pollution-related associations have been observed with cardiac arrest in Rome, Italy,¹⁴⁰ and Indianapolis, Ind,¹⁴¹ but not in Seattle, Wash.^142,143 The mixed results may reflect different PM compositions due to different sources or variations among the methods used.

Table 6 provides a consensus qualitative synopsis based on the expert opinions of the writing group members of the overall level of existing support, linking each surrogate or intermediate cardiovascular outcome with exposures to PM at ambient concentrations, based solely on the database of observational studies.

The molecular events responsible for triggering pulmonary oxidative stress and inflammation, along with the interactions between lung and immune cells, the inhaled PM, and the protective secretions (eg, surfactant, proteins, and antioxidants), are highly complex,^4–6 as reviewed in detail elsewhere.^{290a,290b,414} In brief, size, charge, solubility, aggregation, ROS-producing potential, and chemistry play roles in determining the responses. These include the particle fate (eg, lung clearance versus retention rates), the nature of the PM-cell interactions (eg, immune versus lung cell uptake, host cell responses, and intracellular sequestration/location), and the dose (likely typically a small percentage of inhaled PM) and pathways of potential systemic transmission of PM or its constituents, such as in the circulation [free, intracellular within circulating cells, (lipo)protein-bound] or via lymphatic spread.^{4,5,290a,290b} Because of their nano-scale size, UFPs may directly enter multiple lung cell types via nonphagocytic pathways and adversely affect organelles, such as mitochondria.^6,290a,290b Larger unopsonized fine particles are more typically taken up by phagocytes through interactions with innate immunity receptors such as MARCO (macrophage receptor with collagenous structure) or other scavenger receptors.^5,290a,290b This may in fact be a protective mechanism that sometimes prevents harmful lung inflammation. Certain particle compounds may directly generate ROS in vivo because of their surface chemistry (eg, metals, organic compounds, and semiquinones) or after bioactivation by cytochrome P450 systems (eg, polycyclic aromatic hydrocarbon conversion to quinones).^6,290a,290b A particle surface or anions present on otherwise more inert particles may disrupt iron homeostasis in the lung and thereby also generate ROS via Fenton reactions.²⁹¹ Other PM constituents may do so indirectly by the upregulation of endogenous cellular sources (eg, nicotinamide adenine dinucleotide phosphate [NADPH]) oxidase)^292,293 or by perturbing organelle function (eg, mitochondria) by taken-up PM components.²⁶¹ Particle stimulation of irritant and afferent ANS fibers may also play a role in local and systemic oxidative stress formation.²⁹⁴ Given the rich antioxidant defenses in the lung fluid, secondarily generated oxidization products of endogenous molecules (eg, oxidized phospholipids, proteins) or a reduction in endogenous antioxidants per se may be responsible at least in part for the state of oxidative stress in the lungs (along with instigating the subsequent cellular responses) rather than ROS derived directly from PM and its constituents.

Subsequent to oxidative stress, antioxidant and phase II defenses may be activated (eg, inducible nitric oxide synthase, glutathione) via transcription factor Nrf2-dependent pathways.²⁶¹ When inadequate, pathological oxidative stress can initiate a variety of pulmonary inflammatory responses. For example, ROS in the lungs has been shown to augment the signal transduction of membrane ligand (eg, epidermal growth factor by disrupting phosphatases) or pattern-recognition receptors (eg, toll-like receptors [TLR])^295–299 and/or stimulate intracellular pathways (eg, mitogen-activated protein kinases) that lead to the activation of proinflammatory transcription factors (eg, nuclear factor-κβ) that upregulate expression of a variety of cytokines and chemokines.²⁶¹ Alteration in lung cell redox status may itself stimulate nuclear factor-κβ. Biological components within coarse PM could also directly trigger inflammation (eg, nuclear factor-κβ pathways) by binding to TLR2 or TLR4 receptors or other innate immune pattern-recognition receptors.²⁹⁷ It is also possible that other components of metal-rich PM could instigate inflammatory pathways via TLR activation directly or via the oxidation of endogenous biological compounds that then serve as TLR ligands.³⁰⁰ Finally, there is some evidence that PM can activate inflammatory mitogen-activated protein kinase signaling by angiotensin II receptor–dependent pathways.²⁹⁵ These inflammatory responses can also exacerbate the initial oxidative stress [eg, via upregulation of cellular NAD(P)H oxidase] and thus initiate a positive-feedback cycle.

Available studies support important contributions to pulmonary inflammation from innate immune cells such as neutrophils and macrophages (TNF-α, IL-6), as well as from the adaptive immune system, such as T cells (IL-1, IL-4, IL-6, and IL-10). Although the dominant source of cytokines likely represents the alveolar macrophages and lung epithelial cells, the role of other innate and adaptive immune cells cannot be ruled out.^299,301,302 Recently, myeloperoxidase activity was shown to increase after PM exposure in the same time course of appearance of cellular inflammation (primarily neutrophils) in the lung.³⁰³ Gaseous components such as ozone may also amplify the toxicity of PM.³⁰⁴

In the context of examining the cardiovascular effects of air pollution, it is important to consider the inflammatory mediators that are released from lung cells after contact with PM, because some could conceivably spill over to the general circulation or increase liver production of acute-phase proteins (eg, CRP, fibrinogen). An increase in circulating proinflammatory mediators (eg, activated immune cells, cytokines) could thus serve as a pathway to instigate adverse effects on the heart and vasculature. Numerous experiments have demonstrated increased cellular and inflammatory cytokine content, such as IL-6, IL-1β, TNF-α, interferon-γ, and IL-8, of bronchial fluid and sometimes in circulating blood after acute exposure to a variety of pollutants.^{292,305–311}

Critical roles for the elevations in systemic and pulmonary levels of IL-6 and TNF-α have been observed after PM exposure, typically coincident with pulmonary inflammation.^{292,302,306,309,311–314} There is at least some evidence that the degree of pulmonary inflammation and systemic inflammation (IL-6) correlates with the elevation of systemic cytokines and systemic vascular dysfunction.³¹⁴ In a 4-week inhalation exposure to freshly generated diesel exhaust, IL-6 knockout mice did not demonstrate increased cellular inflammation or TNF-α in bronchial fluid, which implies a role for IL-6.³¹⁵ Consistent with these findings, acute intratracheal exposure to PM₁₀ resulted in an increase in IL-6, TNF-α, and interferon-γ in the bronchial fluid.³¹⁶ However, in this study, IL-6^−/− mice showed roughly the same levels of TNF-α in bronchial fluid as wild-type mice, although interferon-γ was decreased to control values.³¹⁶ The results also suggested that lung macrophages play an important role, because depletion of these cells abolished the increases in some of the cytokines and systemic cardiovascular responses. Although our understanding of the source of IL-6 and TNF-α and their involvement in the systemic inflammatory response after PM exposure remains incomplete, these and other experiments appear to suggest that at least with PM₁₀ particles, alveolar macrophages play a dominant role.^309,314,316

Among remaining uncertainties, the upstream signaling pathway responsible for the recognition of PM components that in turn produce the systemic inflammation has not been fully elucidated³¹⁷; however, there is some evidence with other particulates and experimental models of lung injury that ROS generated by NADPH oxidase or pattern-recognition receptors may modulate some of these responses.^292,299,318 NADPH-oxidase knockout mice demonstrated significantly lower IL-6 and macrophage inflammatory protein-2 responses to collected PM than wild-type mice.²⁹² Extrapulmonary sources may also be involved in promulgating the systemic inflammation. PM_2.5 exposure in a model of diet-induced obesity in C57Bl/6 mice for a duration of 24 weeks resulted in elevations in TNF-α and IL-6. In addition, there were increases in circulating adipokines, such as resistin and plasminogen activator inhibitor-1.³¹⁹ The elevation in cytokines, thought to be derived from adipose sources, in addition to findings of adipose inflammation in that study, raises the possibility of additional systemic nonpulmonary sources of such cytokines.

Numerous in vitro studies have demonstrated activation of ROS-generating pathways by PM incubation, such as NADPH oxidases, mitochondrial sources, cytochrome P450 enzymes, and endothelial nitric oxide synthase in cultured cells or in pulmonary and vascular tissue.^{293,311,320–329} Similar to inflammation, the oxidative stress after PM inhalation may not always stay confined within the lungs.³³⁰ The sources of excess ROS within cardiovascular tissue may include circulating immune cells or cytokines, depletion of defense mechanisms (eg, impaired high-density lipoprotein function), oxidation of lipoproteins or other plasma constituents,³³¹ activation of ANS pathways,²⁹⁴ or circulating PM constituents (eg, soluble metals, organic compounds) reaching the vasculature.²⁶¹ Activation of ROS-dependent pathways modulates diverse responses with far-reaching consequences, including vascular inflammation/activation, atherosclerosis, impaired basal vasomotor balance, enhanced coagulation/thrombosis, and platelet activation.^290b

Recent experiments have indeed confirmed the existence of footprints or markers of oxidative stress within the cardiovascular system in the in vivo context. Acute-exposure studies³³² have shown a relationship between the vascular dysfunction in spinotrapezius microvessels and the release of myeloperoxidase from leukocytes into the vasculature within only hours after the pulmonary instillation of PM.³³² Interestingly, an insoluble particle (TiO₂) induced very similar effects. More long-term studies³³³ have demonstrated that 10 weeks of exposure to PM_2.5 increased superoxide production in response to angiotensin II and resulted in upregulation of NAD(P)H oxidase subunits and depletion of tetrahydrobiopterin in the vasculature. These effects had functional consequences in terms of increases in systemic vascular resistance and BP. In another investigation that involved apolipoprotein E–deficient (ApoE^−/−) fed a high-fat diet, chronic exposure to PM_2.5 exacerbated vascular oxidant stress and promoted atherosclerosis progression.³³⁴ The proatherogenic effects of ambient UFPs³³¹ versus PM_2.5 in genetically susceptible ApoE^−/− mice in a mobile facility close to a Los Angeles freeway have also been compared. Exposure to UFPs resulted in an inhibition of the antiinflammatory capacity of plasma high-density lipoprotein and greater systemic oxidative stress, as evidenced by increased hepatic malondialdehyde and upregulation of Nrf2-regulated antioxidant genes.³³¹

Other experiments²⁹⁴ have suggested that ANS imbalance may play an important role in PM-induced cardiac oxidative stress. Pharmacological inhibition of the ANS could significantly reduce chemiluminescence in the heart after exposure.³⁰³ More recently, an upstream modulator, the transient receptor potential vanilloid receptor-1, within the lung was identified as central to the inhaled CAP-mediated induction of cardiac chemiluminescence.³³⁵ In these studies, capsazepine was able to abrogate ECG alterations in rats during the 5-hour exposure, which suggests that neural ANS pathways are crucial.

Earlier studies using intratracheal instillation of high concentrations of diesel exhaust particles demonstrated the induction of lung inflammation, platelet activation, and increased peripheral vascular thrombosis in both arteries and veins after photochemical injury.^336,337 Thrombosis susceptibility was ascribed to direct passage of the instilled UFPs in the blood, because large polystyrene particles unlikely to cross the lung-blood barrier did not increase peripheral thrombosis. In a subsequent study, a persistent increase in thrombosis susceptibility to diesel exhaust particles was shown after 24 hours, an effect that was mitigated by pretreatment with sodium cromoglycate, which indicates that this response was secondary to histamine release from basophil degranulation.³³⁸ These same effects, however, were mimicked by 400-nm polystyrene particles with a low likelihood of transgressing the pulmonary barrier, which implicates pulmonary release of histamine as a mediator of thrombosis at the later time point. Because histamine was increased in the plasma at 6 and 24 hours after exposure, and diphenhydramine mitigated diesel PM–induced thrombosis at later time points but not at 1 hour, it was hypothesized that additional direct effects of PM constituents reaching the circulation may be responsible for the earliest prothrombotic effects.³³⁹ No increase in circulating von Willebrand factor was observed after instillation of both particles. Finally, pulmonary instillation of carbon nanotubes produced neutrophil lung influx 24 hours later. Circulating platelet-leukocyte conjugates were elevated 6 hours after exposure, whereas procoagulant microvesicular tissue factor activity and peripheral thrombotic potential were increased 24 hours later. Inhibition of P-selectin abrogated these responses, which demonstrates that rapid activation of circulating platelets by the pulmonary deposition of PM plays a vital role.³⁴⁰ This series of studies suggests that release of lung cell–derived mediators (eg, histamine) after several hours along with the more rapid activation of circulating platelets by lung inflammation via P-selectin–dependent processes may mediate distant system prothrombotic effects without necessarily inducing systemic endothelial damage.

In a study using C57BL/6J mice, intratracheal PM₁₀ particles rich in transition metals decreased bleeding, prothrombin, and activated partial thromboplastin times and enhanced the levels of several coagulation factors as well as thrombosis times in response to experimental FeCl₃ injury.³¹⁶ This prothrombotic effect was mitigated in IL-6^−/− and macrophage-depleted mice, which suggests that IL-6, lung macrophages, and pulmonary inflammation are necessary initial steps. It is possible, however, that coarse-particle components (eg, endotoxin) could have been important mechanistically via TLR activation. The effect of fine PM or UFPs per se requires more investigation. Chronic ambient exposure to PM_2.5 has also been shown to increase tissue factor expression in macrophages and smooth muscle cells in atherosclerotic lesions. Complementary in vitro studies with cultured human smooth muscle cells and monocytes demonstrate dose-dependent increases in tissue factor in response to collected ambient particles.³⁴¹ Other findings also support potential procoagulant and thrombotic effects of PM.^342,343 These collective studies suggest that both short- and long-term PM inhalation can enhance thrombotic and coagulation tendencies, potentially via increases in circulating histamine and inflammatory cytokines and/or activated white cells and platelets. The plausibility of these pathways is supported by the well-recognized cross talk between inflammation and thrombosis.³⁴⁴ Potential additional roles for UFPs or soluble constituents that reach the circulation and directly enhance platelet aggregation or systemic oxidative stress (thus activating the endothelium and blunting platelet-derived nitric oxide) require more investigation.

Early animal studies suggested small or inconsistent effects of PM on BP,^345–347 sometimes dependent on the season³⁴⁸ of exposures. A potential explanation may be variations in experimental protocols, including differences in the delivery, duration, and composition of exposure and the methods used to measure BP. Moreover, PM by itself may represent a relatively weak stimulus but may act more robustly in concert with other predisposing factors to affect BP. Sun et al³³³ recently demonstrated a significant interactive effect of fine-CAP exposure with the vasoconstrictor angiotensin II in rats. Preexposure to PM_2.5 for a 10-week period resulted in enhancement of its prohypertensive response measured continuously by intra-arterial radiotelemetry. The exaggerated BP elevation was accompanied by endothelial dysfunction, including blunted endothelium-dependent vasodilation and enhanced vasoconstrictor reactivity, along with upregulation of NAPDH oxidase and Rho-kinase–signaling pathways. In vitro exposure to UFPs and PM_2.5 was also associated with an increase in Rho-kinase activity, phosphorylation of myosin light chain, and myosin phosphatase target subunit. Pretreatment with the nonspecific antioxidant N-acetylcysteine and Rho-kinase inhibitors prevented these responses, which suggests an ROS-mediated mechanism for particle-mediated effects on vascular smooth muscle constriction. Further studies corroborated the role of exaggerated Rho-kinase pathway activity in potentiating the hypertensive response to angiotensin II in mice exposed to PM_2.5.³⁴⁹ Moreover, particle exposure augmented angiotensin-mediated cardiac hypertrophy and collagen deposition. Blockade of Rho-kinase abolished these effects. These responses suggest that chronic PM_2.5 exposure disrupts normal vascular homeostasis and vasoactive mediator balance through ROS-dependent mechanisms in a manner that sensitizes the vessel toward vasoconstrictors. Activation of RhoA/Rho-kinase signaling pathways appears to play an important mechanistic role.

In conscious canines with implanted BP catheters, systemic arterial BP increased and baroreceptor sensitivity was rapidly altered over a few hours during CAP exposure.³⁵⁰ Interestingly, α-adrenergic antagonism abrogated the responses. The findings support a mechanistic role for acute activation of the sympathetic nervous system by inhaled particles. In a study with Wistar-Kyoto male rats, CAP exposure for 4 days upregulated ET-A receptor expression in the heart. This alteration was also weakly correlated with an increase in BP, which suggests a role for enhance ET activity.³⁵¹ PM has also been demonstrated to alter the release of ET-1 and ET-3 from the lungs.³⁵² Elevation in pulmonary vascular resistance and pulmonary arterial pressure, which suggests constriction of the pulmonary vessels, has also been demonstrated in response to respirable carbon black particles.³⁵³ Recently, ultrafine carbon particles were shown to increase BP in spontaneously hypertensive rats 1 to 3 days after a 24-hour exposure.³⁵⁴ This response occurred concomitant with increased ET-1 messenger ribonucleic acid levels in lung tissue and small elevations in plasma renin concentration and angiotensin I and II in the systemic circulation. These findings further support the idea that ET may play a role in cardiovascular responses to PM exposure and suggest that activation of the renin-angiotensin system may also be involved. It is not clear whether the elevated circulating ET levels reflect increased release from the lungs and whether this mediates a systemic vasoconstrictor response. Alternatively, the increase may be more indicative of enhanced vascular tissue activity of these systems. Longer-term exposures of carbon black for 4 weeks in Sprague-Dawley rats has also been shown to significantly increase systolic BP concomitant with increases in serum levels of IL-6 and CRP.³⁵⁵

Finally, in vitro exposure to soluble and insoluble components of UFPs induces constriction in isolated pulmonary arterial rings and activates intracellular signaling pathways such as phosphorylation of extracellular signal–regulated kinase-1/2 and p38 mitogen-activated protein kinase in pulmonary endothelial cells. These effects were antagonized by losartan, and several metal components (copper and zinc) could replicate the responses.²⁹⁵ This suggests a possible role for activation of angiotensin II receptor pathways relevant for the maintenance of vasomotor tone and smooth muscle constriction after inhalation of metal constituents within PM.

In sum, the studies demonstrate that long-term PM exposures over a period of weeks are capable of enhancing vasoconstrictive responsiveness of the vasculature (eg, increased Rho-kinase activity and reduced nitric oxide bioavailability) by inflammatory and ROS-dependent cell-signaling pathways. Shorter-term exposures over several hours to days may lead to vasoconstriction and increased pulmonary and systemic BP by pathways dependent on enhanced ET or angiotensin II signaling. Lung cells may release ET into the systemic circulation and thus increase its systemic activity, or the vascular ET system may be relatively upregulated because of increased ROS or reduced nitric oxide. Activation of the renin-angiotensin system may also occur because of systemic oxidative stress or inflammation or as a consequence of ANS imbalance. The very acute increase in BP that occurs concomitant with the inhalation of particles or within only minutes to hours after exposure appears to be mediated by autonomic imbalance that favors a relative activation of the sympathetic nervous system. No study has evaluated the effect of air pollution on renal sodium handling or long-term pressure natriuresis mechanisms, which are fundamental to the generation of chronic hypertension.

Many early experiments demonstrated the capacity of PM constituents to blunt nitric oxide–dependent dilation and enhance vasoconstrictor tone in ex vivo vascular studies because of excess ROS formation.¹ The first in vivo experiment demonstrated the proatherosclerotic actions of intratracheal PM₁₀ instillation.³⁵⁶ More recently, the pulmonary instillation of several different PM types was shown to rapidly impair microvascular endothelium-dependent vasodilation within days, likely by proinflammatory or ROS-dependent mechanisms (eg, myeloperoxidase).³³² Several animal studies have now demonstrated that long-term exposure to ambient PM_2.5, by use of ambient-exposure facilities without direct pulmonary instillation, not only causes endothelial dysfunction but also accelerates the progression of atherosclerosis. Sun et al³³⁴ demonstrated that exposure of atherosclerosis-prone ApoE^−/− mice to environmentally relevant levels of CAP, derived from regional northeastern PM_2.5, for 6 months in conjunction with a high-fat chow diet potentiated plaque development and heightened vascular inflammation (CD68+ macrophage infiltration and inducible nitric oxide synthase expression) and oxidant stress. The atherosclerotic plaque progression was also accompanied by alterations in vasomotor tone, including decreased endothelium-dependent vasodilation and heightened vasoconstriction to adrenergic stimuli. Importantly, the normalized average PM_2.5 concentration over the entire period was 15.2 μg/m³, which approximates the annual NAAQS. Similar findings were reported in other chronic CAP exposures that involved an ApoE^−/− model.³⁵⁷ However, exposures to a double-knockout model of ApoE-deficient and low-density lipoprotein receptor–deficient mice increased plaque cellularity, reflective of inflammation, but did not enhance plaque burden. It is possible that the atherosclerotic severity of this phenotype precluded the observation of more subtle effects of CAP exposures.

Intratracheal instillation of UFP can acutely impair aortic endothelium-dependent vasodilation.³⁵⁸ Moreover, repeated 10-week-long endotracheal dispersion of UFP carbon black increased atherosclerosis in low-density lipoprotein receptor–knockout mice.³⁵⁹ This occurred without evidence of systemic translocation of particles into the cardiovascular tissues. UFP inhalation by use of exposure facilities has also recently been shown to augment atherosclerosis, perhaps to a greater degree than PM_2.5. When investigating the effects of different PM size fractions, Araujo et al³³¹ compared the proatherogenic potential of exposure over 40 days to ambient particles <0.18 μm versus PM_2.5 in ApoE^−/− mice. UFPs caused more adverse cardiovascular responses (eg, systemic oxidative stress, impaired high-density lipoprotein function) and greater potency in accelerating atherosclerotic lesion formation, although PM_2.5 did demonstrate qualitatively similar effects. Recent studies have also demonstrated that PM exposure likely promulgates systemic atherosclerosis by mechanisms that overlap those of other conventional cardiovascular risk factors.³⁶⁰ Intratracheal instillation of PM₁₀ particles caused a rapid impairment in endothelium-dependent vasodilation, stimulation of bone marrow–derived cells, and increased migration of monocytes into atherosclerotic plaques.^361,362 Systemic inflammation (IL-6) was also related to the degree of endothelial dysfunction.³¹⁴ Finally, the most compelling evidence for rapid impairment in nitric oxide bioavailability being directly involved in the origin of PM-induced endothelial dysfunction was demonstrated recently. Both fine-PM and UFM inhalation for only a few hours in normal rats blunted agonist-stimulated nitric oxide production within the microvasculature, measured by direct electrochemical sensors, concomitant with an observed impairment in vasomotor relaxation. Inhibition of myeloperoxidase or NADP(H) oxidase partially restored normal nitric oxide bioavailability and endothelial function, which suggests a role of activation of these endogenous radical-generating enzymes in this biological response.³⁶³

Potentially relevant adverse vascular effects of nonparticulate PM components should not be discounted. There may also exist some synergy between vapor phase, gas, and particle constituents in relation to instigation of cardiovascular responses. Recently,³⁶⁴ it was demonstrated in apoE^−/− mice that whole gasoline engine exhaust over 1 or 7 days increased vascular messenger ribonucleic acid expression of matrix metalloproteinase (MMP)-2 and MMP-9. Levels of ET-1 and ROS were similarly increased. The vascular ROS and MMP-2 elevations were attenuated by tempol. Endothelial receptor antagonism ameliorated the vascular expression of MMP-2, MMP-9, and ROS. In separate experiments, diesel exhaust exposure to rats for 5 hours augmented ET-induced vasoconstriction, potentially via a blunting of ET-B–induced nitric oxide release.³⁶⁵ The findings suggest that exposure to a fresh mixture of PM, gases, and vapors may play a role in rapidly triggering atherosclerotic plaque vulnerability via ROS and ET-dependent upregulation of MMP levels.

Some studies suggest that predisposed animals may be more susceptible to air pollution–mediated vascular dysfunction. Diesel exhaust particles delivered by intraperitoneal injection impaired nitric oxide–dependent vasodilation only in apoE^−/− mice with atherosclerosis and not in healthy control animals.³⁶⁶ Aortas from prediabetic rats were found to be more susceptible to repeated exposures to oil combustion particles in causing noradrenergic-mediated constriction and impaired endothelium-dependent vasodilation.³⁶⁷

Taken together, the available studies suggest that short- and long-term particle exposures (including PM₁₀, PM_2.5, and UFP) can impair conduit and resistance arterial endothelium-dependent vasodilation. Chronic exposures have been shown to be capable of promoting atherosclerosis progression and enhancing plaque vulnerability. The underlying mechanisms likely involve vascular sequelae of systemic inflammation (due to interactions with innate immune cells and cytokines) or exaggerated oxidative stress pathways. Excess vascular ROS and inflammation will impair endogenous vasodilator bioavailability (eg, nitric oxide), enhance vasoconstrictor tone (eg, ET), and chronically activate multiple intracellular pathways that promote atherosclerosis.^368–370

Some of the earliest indications of systemic effects of PM came from ECG studies in rats.³⁷¹ In general, reductions in several measures of HRV have been shown.^372–376 Most of the recent research has focused on exploring the roles of susceptibility and exposure characteristics. Decreases in heart rate and HRV indices have been reported to be pronounced in senescent mice, which indicates that aging may be a susceptibility factor.³⁵³ Using an anesthetized model of postinfarction myocardium sensitivity, Wellenius and colleagues³⁷⁷ did not demonstrate an effect of 1 hour of CAP exposure on heart rate or spontaneous ventricular arrhythmias. In contrast, in a post-MI heart failure model in Sprague-Dawley rats, diesel exhaust emissions reduced HRV in both healthy and heart failure groups and increased the incidence of premature ventricular contractions. Studies in mice have also indicated a potential role for transition metals and nickel in HRV alterations³⁷⁶ and provide initial clues on the PM components that could influence autonomic tone.⁴⁸

Some beginning insight into the neural pathways involved has been reported recently. PM-induced ECG changes in rats were shown to be prevented by inhibiting the transient receptor potential vanilloid receptor in the lungs. This suggests that the relevant neural mechanism that leads to alterations in HRV or heart rhythm may be induced by activation of receptor-mediated autonomic reflexes in the lung.³³⁵ Circulating particle constituents or inflammatory mediators interacting with myocardial ion channels or electrophysiology did not appear to be a pertinent mechanism, at least in these studies.³³⁵ However, it is unknown whether similar mechanisms can account for the HRV changes observed in humans, and a more detailed understanding of the anatomic pathways involved is required. Finally, it remains unclear whether the changes in cardiac HRV are actually caused by or merely illustrate an underlying alteration in ANS balance. Experiments that clearly define the direct contribution of sympathetic and parasympathetic nervous system activities (eg, microneurography, norepinephrine spillover rates, or autonomic receptor or ganglionic blockade) are needed.

PM exposure can increase experimental infarct size and potentiate myocardial ischemia and arrhythmias in experimental MI models. Relatively high concentrations of intratracheal UFP instillation induced pulmonary inflammation and doubled MI size in mice.³⁵⁸ Conscious dogs exposed to fine CAP for several days experienced greater ST-segment changes during transient coronary artery occlusion.³⁷⁸ These studies suggested that particulate-related changes in myocardial blood flow may be responsible, a hypothesis recently supported by experiments in chronically instrumented dogs exposed to fine CAP before transient occlusion of the left anterior descending artery. PM exposure was associated with a small but significant decrease in total myocardial flow, especially in the ischemic zone, and increases in coronary vascular resistance without an alteration in rate-pressure product.³⁷⁹ The abnormalities were inversely related to PM mass, particle number, and black carbon concentration.

Exposure to residual oil fly ash increases arrhythmia frequency in rats with preexisting premature ventricular complexes, which suggests that PM sensitizes ischemic myocardium to abnormal automaticity³⁷²; however, CAP had no effect in rats.³⁸⁰ Nevertheless, the data suggest that PM exposure may potentially be capable increasing the sensitivity of the myocardium to ischemia, likely by impairing myocardial blood flow and perfusion. In theory, this could play a role in enhancing the propensity for ventricular arrhythmias.

Recently, Sun et al³¹⁹ exposed C57BL/6 mice fed high-fat chow to fine CAP or filtered air for 24 weeks. Mice exposed to PM_2.5 exhibited marked worsening of whole-body insulin resistance, systemic inflammation (increased IL-6 and TNF-α), and higher levels of adipokines, such as resistin and plasminogen activator inhibitor-1. PM_2.5 increased visceral adiposity and inflammation (F4/80⁺ cells), with stromal vascular cells expressing higher TNF-α and IL-6 and lower IL-10 levels. Exposure also induced insulin-signaling abnormalities and reduced phosphorylation of Akt and endothelial nitric oxide synthase in aortic tissue, accompanied by abnormalities in vascular relaxation to insulin. Additionally, there was evidence that PM_2.5 exaggerated adhesion of monocytes in mesenteric microvessels, culminating in accumulation in visceral adipose. These intriguing findings suggest that longer-term exposure to PM air pollution may promote the chronic development of insulin resistance, obesity, and the metabolic syndrome.

Controlled human exposure studies have measured the effects on circulating inflammatory markers such as CRP, IL-6, and TNF-α. In many of these single-episode short-term exposures, no overt changes in plasma cytokine levels were observed after CAP^381–383 or diesel exhaust.^{345,384–386} Similarly, CRP levels have not consistently been found to increase in the time frame and context of most of these studies.^{313,384–386}

However, there have also been some positive findings. Increases in IL-6³¹³ and TNF-α 24 hours after exposure to diesel exhaust in healthy adults have been reported. High levels of ambient particles can stimulate the bone marrow to enhance the release of neutrophils, band cells, and monocytes into the circulation, which causes a cellular inflammatory response.^387,388 Some controlled-exposure studies corroborate the existence of a cellular proinflammatory response that manifests as increases in circulating white blood cell or immune cell counts. In 1 study, increased peripheral basophils in healthy older adults were noted 4 hours after a 2-hour exposure to fine CAP.³⁸⁹ In a similar study, increased white blood cell counts were observed in healthy young adults 12 hours after exposure.³⁸¹ Recently, investigators observed an increased in total white blood cell and neutrophil levels immediately after a 2-hour exposure to CAP in downtown Toronto, Ontario, Canada.³⁹⁰ Conversely, decreases in blood monocytes, basophils, eosinophils, and CD54 and CD18 adhesion molecule expression on monocytes after exposure to ultrafine carbon (10 to 50 μg/m³) among exercising asthmatic individuals and healthy adults have also been reported.³⁹¹ The authors suggested in the latter study that these results may represent the sequestration of these cells in tissue compartments such as the lung or vasculature, where there may be selective expression of the corresponding receptors for these ligands.³⁶² However, other recent human clinical studies have found no association between peripheral blood cell counts and exposure to fine PM or UFPs such as zinc oxide,³⁹² ultrafine carbon,³⁹³ or diesel exhaust.^313,384,385

More subtle, yet physiologically relevant or functional proinflammatory changes may be overlooked by the measurement of circulating cytokines or cell counts alone in human studies. Peretz et al³⁹⁴ recently evaluated gene expression using an expression array in monocytes after 2 hours of exposure to diesel exhaust. Although initially a small study, 10 genes involved in the inflammatory response were modulated in response to exposure (8 upregulated, 2 downregulated). These findings will need to be reproduced in larger studies and raise the possibility that functional changes in inflammatory cells may occur without discernible changes in their levels in the peripheral circulation.³⁹⁴

In sum, the findings from controlled human exposures do not demonstrate a robust inflammatory response; however, they have been limited by the fact that they are, by necessity, of short duration and relatively low concentration. Additionally, the results do not preclude an effect of higher exposures, the presence of more subtle responses, or alterations in other cellular inflammatory pathways not measurable by circulating markers.

The demonstration of systemic oxidative stress is difficult in human studies. Nonetheless, a few studies have reported positive findings. These include an increase in urinary excretion of free 8-iso-prostaglandin-2α among healthy adults after a 4-hour exposure to concentrated wood smoke³⁹⁵ and an increase in plasma antioxidant capacity 24 hours after a 1-hour exposure to diesel exhaust in a group of healthy volunteers.³¹³ The investigators speculated that systemic oxidative stress after exposure may have been responsible for this upregulation in antioxidant defense.³¹³ Other investigators³⁹⁴ have observed significant differences in expression of genes involved in oxidative stress pathways due to diesel exhaust exposure. Bräuner et al¹⁶⁷ recently investigated the effect of ultrafine traffic particles on oxidative stress–induced damage to DNA in healthy young adults exposed to low concentrations of ambient urban particles (PM_2.5 and PM_10–2.5 mass of 9.7 and 12.6 μg/m³, respectively) in an exposure chamber above a busy road with high traffic density. The authors observed increased levels of DNA strand breaks and formamidopyrimidine-DNA glycosylase sites in monocytes after exposure to PM but no changes in the DNA repair enzyme 7,8-dihydro-8-oxoguanine-DNA glycosylase. Similar to their previous findings with ambient levels,¹⁶⁸ the results suggest that short-term exposure to UFPs may result in damage to DNA. This may occur through oxidative stress pathways, although there was no increase in messenger ribonucleic acid levels in heme oxygenase-1, a gene known to be regulated by Nrf2, a transcription factor regulated by oxidative stress.³⁹⁶ Moreover, more recent observations by the same investigators failed to demonstrate significant biomarker signals for lipid or protein oxidative damage after similar near-roadway exposures.¹⁷⁸ Although not entirely consistent, the available studies demonstrate that acute exposure to PM, perhaps even at ambient levels, may be capable of inducing acute systemic oxidative stress in human subjects under certain circumstances. The assays used to assess the footprint of systemic “oxidative stress” or damage may also play a significant role in the results.

Several new studies of controlled human exposure have evaluated the effects of PM on hemostatic markers (eg, factor VII, fibrinogen, platelet count, D-dimer, and von Willebrand factor). Although some of these studies have not observed changes after acute exposures,³⁹² others have reported increases in fibrinogen levels at 8 to 24 hours after exposure to CAP.^381,397 Mills and colleagues^384,385 recently demonstrated a significant effect of diesel exhaust on fibrinolytic function in response to intermittent exercise both in healthy men and in men with coronary heart disease. In both groups of volunteers, bradykinin-induced release of tissue plasminogen activator was observed to decrease compared with filtered air at 6 hours after exposure to diesel exhaust. These perturbations in tissue plasminogen activator release did not persist 24 hours after exposure.³¹³ In a randomized, controlled crossover study involving “at-risk” metabolic syndrome patients, no changes in plasminogen activator inhibitor-1 were noted over a 24-hour duration; paradoxically, a decrease in von Willebrand factor was noted in this study.³⁹⁸ In a similar experiment conducted in healthy adults, diesel exhaust had no effect on D-dimer, von Willebrand factor, CRP, or platelet counts compared with filtered air up to 22 hours after exposure.³⁸⁶ Other investigators³⁹⁵ recently evaluated the effect of wood smoke on markers of coagulation, inflammation, and lipid peroxidation in young healthy subjects. Serum amyloid A and the ratio of factor VIII to von Willebrand factor, an indicator of an increased risk of venous thromboembolism, were increased at 4 hours after exposure.³⁹⁵ Samet et al³⁸³ reported an association between various coagulation markers and exposure to ultrafine, fine, and thoracic coarse CAP among healthy young adults. Although exposure to coarse CAP did not result in significant changes in hemostatic variables, the overall trend suggested a prothrombotic effect. Exposure to UFPs increased D-dimer levels, whereas fine-CAP effects tended to increase fibrinogen, similar to previously reported findings.³⁸¹

The measurement of blood levels of coagulation factors or biomarkers of thrombosis could potentially miss a relevant biological effect at the vascular wall. Recently, ex vivo thrombus formation was assessed by use of the Badimon chamber after controlled exposures to dilute diesel exhaust in healthy volunteers.³⁹⁹ This protocol measures thrombus formation in native (nonanticoagulated) whole blood triggered by exposure to a physiologically relevant substrate, under flow conditions that mimic those found in diseased coronary arteries. It may therefore provide a superior estimate of actual in vivo conditions related to thrombosis potential. Interestingly, dilute diesel exhaust exposure increased thrombus formation within 2 hours, in association with increased platelet activation (ie, increased circulating platelet-monocyte aggregates and soluble CD40 ligand). Taken together, these new studies have provided additional evidence that short-term exposure to PM at near-ambient levels may have small yet potentially significant effects on hemostasis in humans. Whether direct interactions of circulating PM constituents with platelets, activation of platelets due to lung inflammation or secondary to elevated systemic cytokine levels, or an increase in procoagulant factors (eg, fibrinogen) as an acute-phase response to inflammation (or a combination of these pathways) is responsible warrants attention in future studies.

Although several studies have evaluated the BP response to acute exposures, many inconsistencies in results have been reported.⁴⁰⁰ This must be considered in the context that BP was not the primary outcome of interest in most studies, nor was it typically assessed with adequate sophistication. In one of the earliest studies, PM_2.5 increased systolic BP in healthy subjects but decreased it in asthmatic individuals.⁴⁰¹ Three other controlled studies did not report changes among healthy adults.^345,402,403 However, in a more detailed reanalysis of the changes in BP during the actual period of exposure to CAP plus ozone, Urch et al⁴⁰⁴ found a significant increase in diastolic BP of 6 mm Hg. The magnitude of response was associated with the concentration of organic carbon within PM_2.5.⁴⁰⁵ Recent follow-up studies redemonstrated an acute prohypertensive response during the inhalation of CAP in 2 separate cities.³⁹⁰ The PM_2.5 mass during exposure and decreases in several HRV metrics were associated with the magnitude of the short-lived diastolic BP elevation. This suggested that the most plausible mechanism for this acute response was CAP-induced ANS imbalance that favored sympathetic over parasympathetic cardiovascular tone. Whether this reaction occurred because of a generalized stress response, as a consequence of specific soluble PM constituents directly altering central nervous system activity, or via altered ANS reflex arcs due to the interaction of inhaled particles with lung receptors/nerve endings remains to be elucidated.

The effect of inhaled particulates on BP has also been investigated in several other recent controlled human exposure studies. Two new studies assessed BP changes after a 1-hour exposure to diesel exhaust. Mills et al³⁸⁴ found a 6-mm Hg increase in diastolic BP 2 hours after exposure, which was of marginal statistical significance (P=0.08); however, this trend did not persist for 24 hours,³⁸⁴ nor was it found among patients with coronary artery disease.³⁸⁵ The available data to date suggest that short-term exposure to PM_2.5 or diesel exhaust is capable in certain circumstances of rapidly raising BP. The most consistent and largest effects were seen concomitant with the inhalation of particles. Thus far, the most likely mechanism for such rapid hemodynamic responses appears to be ANS imbalance. However, it is possible that reductions in nitric oxide bioavailability that modulate basal arterial tone toward vasoconstriction or increases in ET among other hemodynamically active molecules (eg, angiotensin II) also play a role in some circumstances.

The first controlled human exposure study related to vascular function reported that CAP plus ozone exposure caused acute conduit arterial vasoconstriction in healthy adults.¹ Endothelium-dependent and -independent vasodilation remained intact. Recent follow-up experiments determined that PM_2.5, not ozone, was responsible for the adverse vascular effects. However, in these subsequent and larger experiments, fine-CAP exposure did prove capable of diminishing conduit artery endothelium-dependent vasodilation 24 hours (but not immediately) after exposure.³⁹⁰ Postexposure PM_2.5 mass and TNF-α level were both associated with the degree of endothelial dysfunction, which suggests that systemic inflammation induced by higher levels of particles was likely responsible. Finally, the CAP-induced endothelial dysfunction occurred during exposures in Toronto, Canada, but not Ann Arbor, Mich, which suggests that the composition of the particles is probably an important determinant of the vascular responses.

An acute alteration in vascular function/tone after short-term controlled PM air pollution exposure was corroborated recently.⁴⁰⁶ In 27 adults (10 healthy adults and 17 with the metabolic syndrome), a 2-hour exposure to dilute diesel exhaust caused a dose-dependent constriction of the brachial artery and elevation in plasma ET level without impairing endothelium-dependent vasodilation. Contrary to the hypothesis that metabolic syndrome patients would show greater effects, vasoconstriction was greater in magnitude among the healthy participants. In an additional study, 2-hour exposure to UFPs composed of elemental carbon impaired peak forearm blood flow response to ischemia 3.5 hours later. There were no other vascular changes or alterations at other time points. BP was also not affected.⁴⁰⁷

Several recent studies have also shown that dilute diesel exhaust can impair peripheral resistance vessel responses to acetylcholine, bradykinin, and nitroprusside 6 hours after exposure.³⁸⁴ The blunted responses to acetylcholine persisted for 24 hours in healthy adults.³¹³ In contrast, bradykinin and sodium nitroprusside–mediated vasodilation and bradykinin-induced acute plasma tissue plasminogen activator release were not altered 24 hours later. In subsequent studies, patients with stable coronary artery disease exposed to dilute diesel exhaust for 1 hour during intermittent exercise demonstrated reduced bradykinin-mediated tissue plasminogen activator release; however, microvascular endothelial function was not impaired.³⁸⁵ This may be related to some degree of preexisting endothelial dysfunction in these patients. However, exercise-induced ST-segment depression and ischemic burden were significantly greater during diesel compared with filtered air exposure. These important findings experimentally highlight that PM air pollution exposure can trigger, or augment existing, myocardial ischemia extremely rapidly (in fact, concomitant with exposure). Reduced coronary flow reserve (that was not observed or resolved at the time of the postexposure brachial artery studies) due to rapid alterations in coronary microvascular function may have contributed to the acute myocardial ischemia. Alternatively, acute ANS imbalance induced by diesel exhaust inhalation may have acutely altered coronary tone and impaired myocardial perfusion.

In a study that exposed healthy young adults to 100 μg/m³ of diesel exhaust for 2 hours,³⁶⁴ it was recently demonstrated that this air pollution mixture acutely raised plasma ET-1 and MMP-9 expression and activity within 30 minutes. These results corroborate the animal data that even short-term exposures can rapidly alter factors, such as MMP activity, that are mechanistically linked with causing atherosclerotic plaque disruption (and thus acute MI). The increase in ET levels also corroborates previous studies⁴⁰⁶ that showed that diesel exhaust can acutely affect important endogenous regulators of vasomotor tone.

Controlled air pollution exposures have not always been shown to impair endothelial function or vasomotor tone. Despite an increase in exhaled 8-isoprostane concentrations that suggested pulmonary oxidative stress, fine CAP did not affect brachial flow–mediated dilation or basal diameter in northern Scotland exposures.³⁸² However, the PM_2,5 consisted of relatively inert ambient sea-salt particles and was extremely low in combustion-derived sources. This is in contrast to the particle chemistry in the investigators’ previous diesel exposure studies that showed positive findings.^408,409 Moreover, 24-hour exposure to ambient pollution shunted into a chamber next to a busy street did not impair microvascular endothelial function in 29 healthy subjects, as assessed by digital tonometry.¹⁷⁸ This exposure to near-roadway ambient air, which consisted of ambient UFP and PM_2.5, did not alter biomarkers of inflammation, hemostasis, or protein and lipid oxidation. The authors speculated that the relatively low concentrations of UFP numbers and PM mass or the young, healthy status of the subjects could explain the null findings. Taken together, these studies suggest that brief PM exposure can trigger conduit arterial vasoconstriction, possibly in relation to increased ET activity or augmented sympathetic ANS tone. Under certain circumstances, conduit and resistance arteriole endothelium-dependent vasodilation can also be impaired within a few hours. This abnormality is more likely due to reduced nitric oxide bioavailability as a consequence of systemic proinflammatory and oxidative responses; however, alternative mechanisms and endogenous vasoactive pathways have not been fully explored. It is also apparent that the composition, source, and concentration of pollution, along with the susceptibility of the human subjects, play important roles in determining the vascular effects of acute air pollution exposure.

The results of several new controlled human exposure studies provide limited evidence to suggest that acute exposure to near-ambient levels of PM may be associated with small changes in HRV. There are at least 4 studies to support this. In the first study, healthy elderly individuals experienced significant decreases in HRV immediately after exposure.²³³ Some of these changes persisted for at least 24 hours. Gong et al⁴¹⁰ studied healthy and asthmatic adults exposed to coarse CAPs with intermittent exercise. HRV was not affected immediately after the exposure but decreased in both groups at 4 and 22 hours after the end of the exposure; greater responses were observed in nonasthmatic individuals.⁴¹⁰ In another study, healthy elderly subjects and patients with chronic obstructive pulmonary disease were exposed to approximately 200 μg/m³ CAP and filtered air for 2 hours with intermittent mild exercise. HRV over multihour intervals was lower after CAP than after filtered air in healthy elderly subjects but not in subjects with lung disease. A significant negative effect of CAP on ectopic heartbeats during or after CAP exposure relative to filtered air was noted in the healthy subjects, whereas the group with pulmonary disease experienced an improvement during or after CAP relative to filtered air.³⁸⁹ Other investigators recently compared the effects of 2-hour exposures with intermittent exercise to ultrafine (average concentration 47 μg/m³), fine (average concentration 120 μg/m³), and coarse (average concentration 89 μg/m³) CAP among healthy subjects.³⁸³ In both the ultrafine and coarse studies, a crossover design was used in which each subject was exposed to both PM and filtered air. In the case of the fine-PM study, subjects did not serve as their own control but were exposed to either PM or filtered air. Thoracic coarse fraction CAP produced a statistically significant decrease in the standard deviation of normal-to-normal heart rate 20 hours after exposure compared with filtered air. No statistically significant effects on HRV were observed after exposure to UFPs as measured during controlled 5-minute intervals. However, the authors did observe a significant decrease in the standard deviation of normal-to-normal heart rate after exposure to UFPs based on an analysis of the 24-hour measurements. No differences were reported in HRV with fine-PM exposures. Although some controlled-exposure studies have reported either no acute changes³⁹⁰ or, on occasion, increases in HRV metrics in subsets of individuals,^208,393,401 these studies generally demonstrate that acute PM exposure is capable of reducing HRV. More consistent reductions have been found among older adults (compared with younger subjects or those with lung diseases, who show mixed responses) and perhaps with exposures to larger particles.^233,389 Whether pulmonary ANS reflex arcs are activated by the deposition of PM within the lung or whether other pathways are responsible for these physiological changes in human exposure studies requires more investigation.

Table 7 provides an outline of the level of evidence supporting the generalized intermediary pathways and specific mechanisms whereby PM exposures can be capable of eliciting cardiovascular events. At the molecular level, oxidative stress as a critically important cause and consequence of PM-mediated cardiovascular effects has a sound experimental basis.^{261,290b,294,319,333,334,345–349,351,361–364,411} At the integrated physiological level, the collective body of evidence continues to support the existence of 3 general pathways (Figure 3). Some of these responses, such as systemic inflammation (via pathway 1), likely require antecedent pulmonary oxidative stress or inflammation in order to be initiated. Others, including ANS imbalance (via pathway 2) and PM or its constituents reaching the systemic circulation (via pathway 3), may not. Although PM-associated metals⁴¹² and certain UFPs^{261,413–415} might be capable of translocating into the blood stream, some studies have been negative in this regard.^355,416 Many issues related to this pathway are controversial and require resolution.⁴¹⁶ These include the relevance of the dosages delivered to cardiovascular organs, the consequences of particle constituent modifications after interactions with lung tissue/fluids and plasma components, the means of transport within the circulation (eg, protein bound or within cells),⁴¹⁷ and the time course and ultimate sites of PM sequestration. It is also possible that increases in some vasoactive mediators or molecules with adverse effects on cardiovascular tissue, such as ET-1,^351–354 may occur in the lung and systemic circulation without the need for antecedent lung inflammation. Moreover, the 3 general pathways represent a simplification of complicated biological processes. They may not be mutually exclusive, may overlap temporally, and likely exhibit synergies in causing manifest cardiovascular disease events. Many of the biological pathways are also known to exhibit mutual interactions (eg, inflammation with thrombosis/coagulation and with autonomic function). The pathways are also likely to be principally active at differing time points (eg, more rapid cardiovascular effects of autonomic imbalance than systemic inflammation) and likely vary in importance in relation to different durations of exposure and in causing different cardiovascular sequelae. The chemical characteristics and sizes of inhaled PM may also determine the pathways activated. As opposed to UFPs or some particulate components or chemicals, larger fine and coarse PM are not likely transported into the circulation to any large degree and therefore are more apt to require intermediary pathways to cause extrapulmonary effects. It may also be that surface-bound components may be delivered into the circulation, whereas larger particles themselves serve as a means to deliver the responsible constituent into the pulmonary tree.

The hyperacute physiological responses that occur minutes to hours after PM inhalation are likely mediated principally via pathways 2 and 3. These include ANS-mediated changes (eg, elevated BP, arrhythmias, and vasoconstriction), along with direct effects of circulating PM constituents on platelets (eg, procoagulant and thrombotic changes) and the endothelium (eg, oxidative stress and vasoconstriction). These responses are liable to be the dominant mechanisms responsible for the actual triggering of acute cardiovascular events. Clinically meaningful effects undoubtedly become manifest only in the context of a susceptible patient, typified by the individual with “vulnerable plaque” in the case of acute coronary syndromes or strokes, “vulnerable myocardium” in the context of arrhythmias, or the “vulnerable circulation” in the context of a heart failure patient at risk for circulatory overload. On the other hand, the biological consequences of systemic inflammation, such as activated white cells and elevated cytokines (via pathway 1), typically require longer periods. Their penultimate effect is the induction of a chronic underlying vulnerable milieu that leads to atherosclerotic plaque vulnerability, enhanced coagulation/thrombotic and arrhythmia potential, and impaired basal vasomotor balance. These actions thereby predispose individuals for future cardiovascular events, particularly when they occur in conjunction with traditional risk factors or prompt susceptibility to the acute biological actions (via pathways 2 and 3) of later air pollution exposures.

This hypothetical segregation of the biological effects of PM exposure as acute or chronic and into the broad pathways is artificial. It is useful in the broad context of understanding potential pathways; however, there is no doubt a large degree of overlap among the mechanisms and the timing of physiological responses. This is most aptly conveyed as the influence of “acute on chronic” actions of exposure. For example, the activation of circulating platelets by the pulmonary deposition of particles or lung inflammation (eg, by P-selectin–dependent pathways, histamine, or IL-6) could occur within hours and more rapidly than typical of the other consequences of inflammation (eg, progression of atherosclerosis). In the presence of a vulnerable or eroded coronary plaque due to long-term air pollution exposure, this sudden prothrombotic tendency could instigate an acute ischemic event (alone or in conjunction with other effects of short-term PM exposure via pathways 2 and 3). Furthermore, the epidemiological cohort studies demonstrate a larger relative risk for increased cardiovascular-related mortality than for morbidity.^{72,73,227,274} If this is a true biological response and not simply a statistical phenomenon or a shortcoming of the available data, it not only suggests that exposures are capable of triggering acute cardiovascular events but that PM air pollution may also exaggerate their severity even if they would have otherwise occurred for reasons unrelated to air pollution. Therefore, exposure to PM could also be responsible for promoting fatal over nonfatal events.