Particulate air pollution has been associated with triggering of myocardial infarctions and increased cardiovascular mortality.1 Potential pathways for these effects include increased systemic cytokine-mediated inflammation, endothelial dysfunction, increased thrombosis, decreased plaque stability, and increased arrhythmias.1–3 Previous studies have found that air pollution influences markers of coagulation (such as fibrinogen),4,5 inflammation (such as C-reactive protein),6 and endothelial function (such as intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]).7–9 Elevations in these blood markers, in turn, have been associated with increased risk of adverse cardiovascular events.1,2,9–11 Fibrinogen plays a key role in the clotting cascade, where its conversion to fibrin stabilizes blood clots after injuries. Fibrinogen has procoagulant and proinflammatory properties, and promotes atherothrombosis. C-reactive protein is an acute-phase protein and a general marker for inflammation. ICAM-1 and VCAM-1 are transmembrane proteins that mediate endothelial transmigration of white blood cells in inflammatory response. Air pollution has also been linked to higher tissue factor (F3) and Toll-like receptor 2 (TLR-2) expression.12,13 While TLR-2 is a member of the Toll-like receptor family, which plays an important role in pathogen recognition, tissue factor enables cells to initiate the blood coagulation cascade.
Air pollution effects on cardiovascular disease (CVD) are stronger among subjects with fibrinogen and interleukin (IL)-6 gene variants.14 However, epigenetic modifications may be as important as genetic polymorphisms in CVD pathogenesis.15 Epigenetics refers to potentially mutable marks on the chromosome that influence gene expression. These marks are established mostly in utero and are relatively stable over time. DNA methylation is an epigenetic modification that usually results in silencing of nearby genes. Most DNA methylation occurs in intergenic regions, such as in Satellite, Alu, and LINE-1 repetitive elements. LINE-1 hypomethylation seems to play an important and consistent role in diseases,16,17 and has itself been associated with CVD and survival time.18–20 Risk of stroke has also been related to Alu hypermethylation.21
We focused our investigation on the relative toxicity of various sources (traffic versus coal combustion) and components of air pollutants. We first hypothesized that increased air pollution is associated with changes in fibrinogen, C-reactive protein, ICAM-1, and VCAM-1 levels, and that various sources and components of air pollution affected these outcomes differentially. Although epigenetic control of gene expression is important to DNA repair and cell proliferation, it may also play a key role in inflammatory gene expression.15 Therefore, we also examined effect modification by methylation of LINE-1, Alu, F3, TLR-2, and ICAM-1. Methylation at specific positions within a gene's promoter region may be more important for gene expression than the average methylation of CpG sites in the promoter region.22 Therefore, we investigated the epigene-environment interactions using average and position-specific methylation.
This prospective cohort study included subjects from the Normative Aging Study, an investigation of aging established in 1963 by the US Veterans Administration and enrolling community-dwelling men from the Greater Boston area. Participants were confirmed to be free of known chronic medical conditions and visited the study center every 3 to 5 years to undergo physical examinations. Visits took place in the morning, after an overnight fast and smoking abstinence. Our analyses included 704 subjects whose levels of fibrinogen, C-reactive protein, ICAM-1, VCAM-1, and DNA methylation were measured at least once between 2000 and 2009. Because elevated C-reactive protein levels usually reflect an infection state, we restricted our analyses to participants having levels less than 10 mg/L. Attrition has been less than 1% per year, and is due predominantly to moving from the study area or death. This study was approved by the institutional review boards of all participating institutions.
Ambient particulate concentrations were measured at a central monitoring site (1 km from the medical center). We measured hourly ambient fine particle (PM2.5) concentrations with a Tapered Element Oscillation Microbalance (Model 1400A, Rupprecht and Pastashnick, East Greenbush, NY), and hourly black carbon concentrations using an Aethalometer (Magee Scientific Co., Model AE-16, Berkeley, CA). Occasional missing daily values of black carbon and PM2.5 were estimated using regression models that included previous and subsequent pollutant measurements, meteorologic data, seasonality, and long-term trend. We measured hourly particle number per cm3 (0.007–3 μm) with a Condensation Particle Counter (TSI Inc, Model 3022A, Shoreview, MN). We determined daily sulfate (SO42−) concentrations using a Sulfate Particulate Analyzer (Thermo. Electron Co., Model 5020, Franklin, MA), between 2000 and 2003. Subsequently, SO42− levels were calculated from elemental sulfur, measured by x-ray fluorescence from particle filters. For the year of overlap, we fit a calibration regression, which had an R2 over 0.9, and a slope of 1. We obtained hourly measurements of carbon monoxide (CO), ozone (O3), and nitrogen dioxide (NO2) from local state monitors, taking the mean site values for each gas. Air pollution measured by the monitors is used as a surrogate for urban personal exposure. The median distance of the participant homes from the central site monitoring station was 19.0 km; the medians of the mean distances of the participant homes from the CO, O3, and NO2 monitors were 20.2, 22.3, and 21.4 km, respectively. The appropriate averaging time for the air pollution effect on coagulation, inflammation, and endothelial function is unclear. Previous studies suggest that the effect is spread overall several days,23 months,24 or even years.25 For each pollutant concentration, we considered the short- and intermediate-exposure windows of 4 hours, 24 hours, and 3 to 28 days moving average (MA3 to MA28) preceding each subject's examination.
We collected blood at every visit to measure LINE-1, Alu, and gene-specific methylation, following previous published methods.19 We isolated bisulfite-treated DNA with the Wizard DNA Clean-Up System (Promega, Madison, WI) and measured DNA methylation using highly quantitative analysis based on PCRpyrosequencing. We determined levels of F3, TLR-2, and ICAM-1 methylation at multiple CpG positions within each gene's promoter region and calculated the mean values of the position-specific measurements. The assay quantifying the gene-specific methylation levels was developed by locating the promoters using Genomatix Software (Genomatix Software Inc, Ann Arbor, MI). The degree of methylation was expressed as the percentage of methylated cytosines over the sum of methylated and unmethylated cytosines.
Outcome Blood Markers
At each visit, we measured plasma fibrinogen using MDA Fibriquick, a thrombin reagent. We determined C-reactive protein concentrations with an immunoturbidimetric assay on the Hitachi 917 analyzer (Roche Diagnostics-Indianapolis, IN). We obtained plasma ICAM-1 and VCAM-1 levels using an enzyme-linked immunoabsorbent assay method (R&D Systems, Minneapolis, MN).
Fibrinogen, C-reactive protein, ICAM-1, and VCAM-1 levels were log-transformed to guarantee a normal distribution of the residuals. Subjects had their blood marker levels measured on up to 4 visits. Because most participants had repeated measurements of the outcomes of interest, we analyzed our data using separate mixed models with a random intercept for each subject. This gives unbiased estimates of the fixed effects even when some subjects had only one measurement. We chose the following covariates a priori: age, seasonality, body mass index (BMI), temperature, relative humidity, smoking status, statin use, and diabetes. Seasonality was controlled using sine and cosine terms with a period of 365.24 days. Both sine and cosine were included to allow the regression to estimate both the amplitude of the seasonal cycle and its phase. Using interaction terms, we investigated air pollution effect modification by LINE-1 and Alu methylation on every blood marker of interest, as well as air pollution interactions with F3 modifying fibrinogen, TLR-2 changing C-reactive protein, and ICAM-1 modifying the ICAM-1 and VCAM-1 proteins' concentrations. We dichotomized methylation levels, using the 50th percentile of the distribution as a cut-off point. Because the pattern of 5-methylcytosine distribution in the genome of differentiated somatic cells varies according to cell type, we adjusted for percentages of neutrophil and lymphocyte in differential blood count. Our statistical model investigating epigene-environment interactions was:
- Yij: logarithm of blood marker for subject i at the jth measurement
- Zij: covariates for subject i at the jth measurement
- bi: random intercept
- εij: within-subject errors
We present the subjects' baseline characteristics and the weather distributions in the Table. The study population was elderly, with a mean age of 73.2 years. Air pollution characteristics are reported in eAppendices 1 to 4 (https://links.lww.com/EDE/A545). There were 4 sites for NO2 and CO, and 2 for O3. Spearman correlations between monitors were 0.84 for O3, and ranged from 0.52 to 0.75 for NO2 and from 0.35 to 0.57 for CO. We observed correlation coefficients of 0.72 between black carbon and PM2.5, 0.83 between PM2.5 and SO42−, and −0.43 between particle number and O3. We described the different positions within the promoter region of the F3, TLR-2, and ICAM-1 genes in eAppendix 5 (https://links.lww.com/EDE/A545).
Air Pollution Main Effects
We observed positive effects of traffic-related pollutants on fibrinogen (Fig. 1). An interquartile range increase in exposure (3-day moving average) was associated with a 2.4% (95% confidence interval [CI] = 0.1%–4.81%) increase in fibrinogen for particle number, a 2.6% (0.9%–4.3%) increase for black carbon, a 4.5% (2.6%–6.4%) increase for NO2, and a 2.5% (0.9%–4.0%) increase for CO. In contrast, PM2.5, SO42−, and O3 (which are mostly secondary pollutants) were not associated with fibrinogen. O3 was, however, a predictor of C-reactive protein and ICAM-1 (Figs. 2, 3). A 24-hour interquartile range increase in O3 was associated with a 10.8% (2.2% to 20.5%) increase in C-reactive protein. We found higher ICAM-1 levels after short- and intermediate-term exposures to particle number, PM2.5, and SO42−, and only after intermediate-term exposure to black carbon, NO2, and CO (Fig. 3). We obtained similar associations for VCAM-1 (Fig. 4). ICAM-1 and VCAM-1 were negatively related to intermediate-term ozone exposure. However, after adding particle number to the model, these associations were weakened.
Effect Modification by DNA Methylation Status
Repetitive Elements Methylation
We observed stronger effects of traffic-related pollutants on fibrinogen and C-reactive protein, among subjects with lower LINE-1 or higher Alu methylation status (Fig. 5). While among subjects with higher Alu methylation, an interquartile range increase in NO2 (14 day moving average) was associated with a 6.4% (3.9% to 8.5%) increase in fibrinogen, we observed only a 2.4% (−0.04% to 5.0%) fibrinogen increase among participants with lower methylation. LINE-1 also increased the effect on fibrinogen of SO42−, after 1 to 4 weeks of exposure. The air-pollution effect on ICAM-1 and VCAM-1 was not modified by methylation of repetitive elements status.
The air-pollution effect on fibrinogen was not modified by the mean tissue factor (F3) methylation states. However, when we examined effect modification by position within the promoter region, we observed longer short- and intermediate-term effects on fibrinogen of particle number, black carbon, NO2, and CO among subjects with low F3 methylation status for Position 1 or 5 (Fig. 6). For instance, among the low F3 methylation group for Position 1, an interquartile range increase in NO2 (3-day moving average) was associated with a 6.2% (3.8% to 8.5%) increase in fibrinogen, but only with a 2.9% (0.5% to 5.5%) increase among participants with higher methylation levels. When examining interaction between air pollution and TLR-2 methylation on C-reactive protein, we found greater intermediate-term effects of particle number and NO2, among the low TLR-2 methylation group (Fig. 6). We examined effect modification by ICAM-1 methylation status on ICAM-1 and VCAM-1 levels and did not find any consistent interactions.
As sensitivity analysis, we investigated the epigene-environment interactions using continuous methylation variables; the direction and magnitude of the results did not change. Air pollution and smoking (pack-years or history) were not correlated in our data set. We also examined effect modification by smoking and found no evidence of interactions (results not shown). Because air pollution and methylation are correlated,26 the epigene-air pollution interactions could be the result of a nonlinear effect of air pollution on the blood markers of interest. We checked this possibility by regressing methylation on air pollution and relevant covariates. Because the resulting residuals reflected the methylation variability independent of air pollution, dichotomizing them into low versus high avoids the ambiguity due to effects of air pollution on methylation. When we used them for the interaction term with air pollution, we obtained similar interactions.
Our results suggest that current concentrations of traffic-related air pollutants increase levels of intermediary CVD-related blood markers (fibrinogen, C-reactive protein, ICAM-1, and VCAM-1) after short- and intermediate-term exposures to air pollution (up to one month), and that those responses vary with a person's baseline DNA methylation pattern. Our results support previous findings that showed influence of air pollutants on markers of coagulation, inflammation, and endothelial function.5,7–9,23,27
We observed short- and intermediate-term effects of particle number on ICAM-1 and VCAM-1. This short time window is consistent with previous experimental studies. After 1 hour of diesel particles inhalation in a chamber, volunteers had increased ICAM-1 and VCAM-1 levels in their bronchial lining fluid and tissue.27 After concentrated fine-particle exposure, ICAM-1 levels measured in venous blood increased after 4 hours and 22 hours.28 A similar study showed that after hours of diesel particles exposure, healthy subjects had higher VCAM-1 concentrations in the bronchial mucosa.29 For the other traffic-related pollutants, we observed only intermediate-term effects on fibrinogen, ICAM-1, and VCAM-1. Zeka et al5 also found lagged exposure effects of traffic-related particles (1 week) and black carbon (1 month) on fibrinogen. Higher VCAM-1 have been reported after days of black carbon exposures and higher ICAM-1 after weeks of exposure.7,8 A key finding in our study was the associations with secondary pollutants as well as primary pollutants. ICAM-1 and VCAM-1 were also higher after short- and intermediate-term exposure to PM2.5 and sulfates, which is consistent with a previous study reporting similar PM2.5 and sulfate effects (5-day moving average).30 We also found a short-term ozone effect on ICAM-1 and C-reactive protein. A positive association between C-reactive protein and ozone was previously obtained after 3 days of exposure.6
We found the strongest associations with particle number, which is an assessment of fresh traffic emissions. Black carbon is, however, a mix of fresh local emissions and some aged transported traffic particles. In Boston, black carbon shows daily patterns peaking at 6 AM and a lower peak in the afternoon rush hour, suggesting a predominance of local traffic. However, transported black carbon is also important, as its concentrations were higher when the back trajectory of the air mass over Boston originated in New York,31 indicating some secondary contribution as well. NO2 is a secondary pollutant that reflects both local sources and transported pollution. These differences might explain why the time pattern of the effects of particle number appears to be distinct from that of black carbon and NO2. Even though particle number, black carbon, and NO2 are traffic-related pollutants, they may be markers of something more complex, including the freshness of the particles. PM2.5 signals on fibrinogen were weak, which is concordant with previous studies.5,32 Fine particles are too small to easily settle out by gravity and too large to coagulate into larger particles, which makes them able to stay in the atmosphere for weeks and travel thousands of kilometers before returning to Earth. In Boston, sulfate particles and secondary organic aerosols constitute a large fraction of PM2.5 mass, with a lesser role for traffic particles. SO42− and O3 (which are other secondary pollutants) were not associated with fibrinogen. Hence, our findings on fibrinogen clearly point toward local traffic particles.
We also observed short- and intermediate-term effects of traffic-related particles, PM2.5, and SO42− on ICAM-1 and VCAM-1. These results on adhesion molecules point toward both primary and secondary pollutants. We reported short-term ozone effects on C-reactive protein and ICAM-1. Ozone is a powerful oxidizing agent and is created with high concentration of pollution and daylight UV rays at the Earth's surface. Ozone exposure has been linked to lung dysfunction and respiratory system irritation,33 and the C-reactive protein and ICAM-1 increases indicate acute inflammatory and endothelial responses to short-term exposure.
Our main air pollution findings suggest some plausible biologic mechanisms that could explain some of the exacerbation of CVD morbidity and mortality.2,27 Air pollution exposure may increase systemic cytokine-mediated inflammation and prothrombotic activity. In susceptible people, ultrafine particles were able to provoke alveolar inflammation, with release of mediators capable of increasing blood coagulability.34 Increased plasma viscosity is a potential mechanism explaining why high fibrinogen levels are related to increased CVD risk.3 Similarly, elevated C-reactive protein, ICAM-1, and VCAM-1 levels have been associated with inflammation and cardiovascular risk.10,35 Increase in C-reactive protein may reflect arterial damage from white blood cell invasion and inflammation within the wall due to air pollution exposure, thus inducing cardiovascular events. In normal human arterial tissues, ICAM-1 levels were lower than in atherosclerotic lesions.11 ICAM-1 plays a role in general inflammation and up-regulation in nonendothelial cells, while VCAM-1 may be more involved in distribution in the vascular system.9 This could explain why the ICAM-1 and VCAM-1 signals have slightly different time windows.
We found greater air pollution effects among subjects with high Alu, but low LINE-1, F3, or TLR-2 methylation status. To our knowledge, this is the first study to report evidence of epigene-environment interactions. Our results suggest that DNA methylation may play an important role in inflammatory gene expression and CVD. Knock-out mice for methylation enzymes develop atherosclerotic fatty streaks.36 In the same cohort, LINE-1 hypomethylation was associated with higher VCAM-1 protein levels,19 as well as higher CVD incidence and mortality.20LINE-1 elements, as retrotransposable sequences, have enhanced activity when they are demethylated, which may induce gene transcription dysfunction, and alterations of the genome by insertion or homologous recombination.37LINE-1 expression has been identified as a mediator of ischemic heart damage.18 Repetitive elements become activated during oxidative and cellular stress conditions.38 Interestingly, recent studies have reported a higher risk of cardiovascular events among subjects with low LINE-1, but also with high Alu methylation in peripheral blood leukocytes.20,21Alu and LINE-1 have also showed opposite changes according to season.26 Therefore, LINE-1 hypomethylation and Alu hypermethylation may be distinct responses to oxidative stress and inflammation, and therefore might identify a subset of subjects who have stronger molecular responses to air pollution.
Air pollution effects on fibrinogen were stronger in subjects with lower F3 methylation. F3 expression and fibrinogen increase in response to proinflammatory stimuli, such as those caused by air pollution.4,12 Tissue factor upregulation is found in inflammatory and hypercoagulable states, which are accompanied by some elevations of clotting signals, including fibrin overproduction.39 Tissue factor hypomethylation may participate in pathways leading to increased tissue factor expression and fibrinogen in response to air pollution and therefore reflects a proinflammatory state leading to stronger coagulation responses.
We observed greater air pollution effects on C-reactive protein among participants with lower TLR-2 methylation. TLR-2 plays an important role in activation of innate immunity. Several cytokines and interleukins, including IL-6, assist in this immunity process. Compared with knockout mice for TLR-2, wildtype mice had greater PM2.5 effects on IL-6.13 IL-6 rise has been associated with increase in C-reactive protein, which also plays a role in innate immunity as an early defense system against infections. Our findings suggest that TLR-2 hypomethylation may silence the TLR-2 gene and participate in biologic processes enhancing IL-6 and C-reactive protein after air pollution exposure, and therefore induces a proinflammatory state leading to stronger inflammatory responses.
Our results suggest that Alu, LINE-1, F3, and TLR-2 methylation status interact with several air pollutants. While tissue factor methylation status seems to modify the effects of all traffic pollutants on fibrinogen, we observed effect modification by LINE-1 methylation only with black carbon and sulfate. Alu and TLR-2 methylation status interacted with NO2 and particle number. Particle number represents freshly generated ultrafine traffic particles, while black carbon captures both fresh and aged traffic particles. The finding that the freshest and smallest traffic particles interact with Alu, TLR-2, and tissue factor suggests a different mechanism, perhaps movement of the ultrafine particles into the blood stream. The interactions of LINE-1 methylation with sulfates and black carbon, capturing fewer fresh particles, are probably related to a different pathway. A recent study supports this hypothesis.40 After 4 hours of ultrafine particles exposure, mice had increased thrombin formation, related to tissue factor (extrinsic coagulation pathway). Alternatively, the procoagulant chronic effects of particulate matter could be mediated via factor XII activation (intrinsic coagulation pathway).
While Alu methylation state modified the particle-number effect on fibrinogen and C-reactive protein after the first hours and days of exposure, it increased the NO2 effect on fibrinogen after 2 to 4 weeks. Similarly, F3 methylation interacted with all traffic-related pollutants over the first hours or days, while LINE-1 interacted with black carbon or SO42− after a longer period (1 to 4 weeks) to modify fibrinogen. These time differences indicate possible distinctions in the properties of air pollutants—especially between primary and secondary pollutants, for which a longer averaging time is required to see an interaction, suggesting different biologic mechanisms.
Strengths and Limitations
We prospectively investigated the role of air pollution from various sources on repeated measures of intermediary CVD-related markers. Our findings were not the result of confounding by CVD risk factors, such as age, seasonality, BMI, temperature, relative humidity, diabetes, statin, and smoking status. Smoking is an important predictor of methylation. However, we found no correlation between air pollution and any smoking variable (category or pack-years). Therefore, we believe that smoking is not confounding the air pollution effects seen in our study. With regard to the possibility that the interaction with methylation is really an interaction with past smoking that produced stable changes in methylation patterns, we examined this question using interaction terms with former and current smoking rather than with methylation. None of these associations explained our results. The observed epigene-air pollution interactions were also not the result of a nonlinear effect of air pollution on the blood markers of interest. Our analysis is, however, limited to 5 methylation variables. Methylation of other genes and histone modifications might be important variables to evaluate. To be confounding variables, they would have to be correlated with Alu, LINE-1, F3, or TLR-2 methylation. There are few data to indicate how likely this is. In addition, DNA methylation is more stable than mRNA or protein expression, and therefore represents a somewhat longer time window in system behavior. We also identified positions within promoter regions of F3 and TLR-2 that may play an epigenetic role in coagulation and inflammation.
We were not able to assess personal air pollution exposure, which is likely to be nondifferentially misclassified. Therefore, exposure misclassification for using ambient monitors presumably attenuates our associations. This attenuation should be larger for short-term exposures and for pollution components that have the greatest spatial variability, which are the traffic pollutants. Exposure assignments for ozone and particularly for sulfates are expected to be fairly accurate and reinforce the null effects found on fibrinogen. Fibrinogen, C-reactive protein, ICAM-1, VCAM-1, and DNA methylation are measured directly from blood. Even though these measurements are likely to be accurate, we also expect some nondifferential misclassification of the underlying biologic function. Our cohort consists of elderly men that may be less susceptible than the population they are representing. Loss to follow-up may also make this study population healthier than the initial population. Furthermore, our results may not be generalizable to the general population if the biologic mechanisms are modified by race, sex, and age.
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We thank the Normative Aging Study participants, Tania Kotlov, and Johanna Lepeule.