Skip to main content
NCBI home page
EPA Author Manuscripts logoLink to EPA Author Manuscripts
. Author manuscript; available in PMC: 2021 May 15.
Published in final edited form as: J Toxicol Environ Health A. 2020 May 15;83(9):351–362. doi: 10.1080/15287394.2020.1762814

Effects of ambient ozone exposure on circulating extracellular vehicle microRNA levels in coronary artery disease patients

Hao Chen 1, Yunan Xu 2, Ana Rappold 3, David Diaz-Sanchez 3, Haiyan Tong 3
PMCID: PMC7306136  NIHMSID: NIHMS1598448  PMID: 32414303

Abstract

Exposure to ambient air pollutants such as ozone and particulate matter (PM) is associated with increased cardiovascular morbidity and mortality, but the underlying biological mechanisms have yet to be described. Emerging evidence shows that extracellular vehicle (EV) microRNAs (miRNAs) may facilitate cell-to-cell and organ-to-organ communications and play a role in the air pollution-induced cardiovascular effects. This study aims to explore the association between air pollutant exposure and miRNA changes related to cardiovascular diseases. Using a panel study design, fourteen participants with coronary artery diseases were enrolled in this panel study. Each participant had up to 10 clinical visits and their plasma samples were collected and measured for expression of miRNA-21 (miR-21), miR-126, miR-146, miR-150, and miR-155. Mixed effects models adjusted for temperature, humidity, and season were used to examine the association between miRNA levels and exposure to 8-hour ozone or 24-hour PM2.5 up to 4 days prior. Results showed that miR-150 expression was positively associated with ozone exposure at 1–4 days lag and 5d moving average while miR-155 expression tracked with ozone exposure at lag 0. No significant association was found between miRNA expression and ambient PM2.5 at any time point. β-blocker and diabetic medication usage significantly modified the association between ozone exposure and miR-150 expression where the link was more prominent among non-users. In conclusion, we observed an association between exposure to ambient ozone and circulating levels of EV miR-150 and miR-155. The findings pointed to a future research direction on the miRNA-mediated mechanisms of ozone-induced cardiovascular effects.

Keywords: Air pollutants, PM2.5, ozone, miRNA, beta blocker, repeated measures, mixed effect model

1. Introduction.

Cardiovascular diseases (CVD), specifically ischemic heart disease and stroke, ranks as leading causes of death worldwide (Heron, 2018; WHO, 2018). In the United States, CVD contributes to approximately 31% of all deaths and costs about 580 billion dollars in health care (Heron, 2018; Mozaffarian et al., 2015). Air pollution is recognized as a major risk factor for CVD morbidity and mortality (Collaborators, 2018). Studies have shown that exposure to particulate matter (PM) or ozone is linked with adverse cardiovascular health effects such as coronary artery diseases (CAD), cardiac arrest, and ischemic stroke (Brook et al., 2010; Day et al., 2017; Mirowsky et al., 2017; Nuvolone et al., 2013). However, the mechanisms through which inhalational exposure to air pollutants leads to adverse health effects on the cardiovascular system are not well understood.

Some proposed mechanisms include that air pollutants induce indirect endothelial injury through systemic inflammation (An et al., 2018; Chuang et al., 2007). Pulmonary injury caused by air pollutants may lead to systemic inflammation where pro-inflammatory cytokines and chemokines (interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α)) or inflammatory cells (e.g. macrophage, neutrophils) in the circulation can elicit endothelial cell injury (Chuang et al., 2007; Ghio et al., 2000; Salvi et al., 1999; van Eeden et al., 2001). Oxidative stress in the lung induced by exposure to air pollutants can also activate transcriptional factor (e.g., nuclear factor (erythroid-derived 2) -like 2-related factor (NRF2) and aryl hydrocarbon receptor (AhR)) dependent regulatory pathways leading to atherosclerosis and ultimately CVD (Lawal, 2017). Some research has suggested that toxicants found in air pollution may enter the blood stream through alveoli, causing direct toxicity to the cardiovascular system (An et al., 2018). Ultrafine particles (UFPs) with a mean aerodynamic diameter < 100 nm may translocate into the blood circulation through the alveoli, reaching the heart and peripheral blood vessels (Furuyama et al., 2009).

Recent research has provided new insights into the role of miRNAs (miRNA) in air pollution induced cardiovascular effects. miRNAs are a family of small (~22 nucleotides), single-stranded, noncoding RNAs that are important in post-transcriptional regulation of gene expression in cells (Fernandez-Hernando and Suarez, 2018; Li et al., 2019). Changes of miRNA profile are common in cardiovascular diseases and are indication of disruption of gene function regulation and endothelial homeostasis (Pereira-da-Silva et al., 2018). Extracellular vehicles (EV) in the circulation can serve as a vehicle for cell-to-cell communication where contents in the EV, such as RNAs, lipids, proteins, and miRNAs, can carry information between cells; for example, from lung cells to endothelial cells in heart and peripheral blood vessels (Kao and Papoutsakis, 2019). Altered expression levels of certain miRNA targets in the circulation might serve as a possible mechanism of cardiovascular effects induced by the pulmonary exposure to air pollutants.

Epidemiological studies have shown that some populations are more susceptible to air pollution, including children, the elderly, the obese, and those with underlying lung and heart diseases (Medina-Ramon and Schwartz, 2008; Mirowsky et al., 2017). A few studies have demonstrated that EV miRNA changes in the circulation are associated with ambient PM exposure in the older population (Louwies et al., 2016; Rodosthenous et al., 2016; Rodosthenous et al., 2018). For example, long-term exposure to ambient levels of PM2.5 has been associated with increased levels of several miRNA targets found in the circulation in the elderly (mean age 76 yrs.) (Rodosthenous et al., 2016). These miRNA changes in response to long-term ambient PM2.5 were linked with DNA methylation patterns that could modify cardiovascular endpoints such as systolic blood pressure (Rodosthenous et al., 2018). However, the role of miRNAs in air pollution-induced cardiovascular effects is still not well understood and studies in multiple air pollutants (e.g. PM2.5, ozone) or susceptible populations are sparse.

Previous work has shown that exposure to ambient ozone is associated with changes in plasma metabolites, inflammation, fibrinolysis, and endothelial cell function among CAD patients (Breitner et al., 2016; Mirowsky et al., 2017). In this study, we aimed to determine whether exposure to ambient PM2.5 or ozone is associated with expression levels of EV miRNA in the circulation among CAD patients. Five miRNAs (miR-21, miR-126, miR-146, miR-150, and miR-155) implicated in normal physiology and the pathogenesis of cardiovascular diseases (Bronze-da-Rocha, 2014; Cao et al., 2016; Cheng and Zhang, 2010; Li et al., 2019; Paterson and Kriegel, 2017) were selected as representative miRNAs that may change in response to ambient air pollution exposure.

2. Materials and methods

2.1. Study design and sample collection

The study design had been detailed previously (Mirowsky et al., 2017). Briefly, subjects who were qualified to enroll should have a stable clinical status, documented coronary artery disease (>75 occlusion in one major coronary vessel), a stable medication regimen over 3 months prior to enrollment, and an electrocardiogram demonstrating normal sinus rhythm. In total, 15 subjects aged between 40 and 75 years old who had previously undergone a cardiac catheterization were recruited to this panel study. To minimize day-of-week effects, each study session was always on the same 2 days of the week for up to 10 sessions. Fasting blood samples were collected on the second visit day of each session. Plasma samples were stored at −80°C prior to miRNA extraction and analysis. Demographics, information on current health status (e.g. body mass index (BMI), blood pressure), current medication usage (e.g. beta blockers, statins, diabetic medications), and past health status (e.g. previous myocardial infarction, smoking history, hypertension) was also collected. Written informed consent was given to all participants prior to enrollment, and the study was approved by the Duke University Institutional Review Board, the University of North Carolina at Chapel Hill Institutional Review Board, and the EPA Human Protocols Office.

2.2. Air pollution measurement

As described previously, data for ozone and PM2.5 levels were obtained from central air monitoring stations close to the EPA Human Studies Facility (HSF) (Mirowsky et al., 2017). Daily 24-hour average of the PM2.5 data were calculated from hourly pollutant data averaged between 9 AM to 8 AM. Daily 8-hour average levels of ozone were used for the analysis. Concentrations were obtained for each visit session, as well as for 4 days prior.

2.3. Extracellular vehicles miRNA extraction

EV miRNA were extracted and lysed from plasma samples using exoRNeasy Serum/Plasma Midi Kit (Qiagen) according to the manufacturer’s instruction. Briefly, exosomes from the plasma samples were isolated by membrane-affinity binding and lysed using QIAzol. Total RNA was recovered from chloroform-induced phase separation. Total RNA products were washed, and miRNA was isolated by membrane-affinity binding method. The concentration of miRNA was quantified by using a NanoDrop spectrophotometer and quality was checked by using Bioanalyzer system (Agilent).

2.4. RT-qPCR

Twenty nanograms of miRNA product was reverse transcribed using a TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher) according to the manufacturer’s instruction. Briefly, exosomes from the plasma samples were isolated by membrane-affinity binding and lysed using QIAzol. Total RNA was recovered from chloroform-induced phase separation. Total RNA products were washed, and miRNA was isolated by membrane-affinity binding method. The concentration of miRNA was quantified by using a NanoDrop spectrophotometer and quality was checked by using Bioanalyzer system (Agilent). For PCR reaction, 5 μl cDNA (1:10 dilution) was used with 15 μl PCR reaction mix including target primers (hsa-miR-21–5p, hsa-miR-126–5p, hsa-miR-146–5p, hsa-miR-150–5p, and hsa-miR-155–5p) in the TaqMan™ Advanced miRNA Assay (ThermoFisher). Quantitative PCR was run on the StepOnePlus™ Real-Time PCR System (ThermoFisher) and data were normalized relative to the endogenous control has-miR-192–5p (ThermoFisher) using the double delta Ct method (Schmittgen and Livak, 2008). The sample with highest delta Ct value for each miRNA target was selected as the caliber for the delta-delta Ct calculation.

2.5. Statistics

Each subject completed from 1 to 10 repeated measurements. Data were analyzed using SAS 9.4 software using a Generalized Linear Mixed model with random subject effects that diminished the need to adjust for subject characteristics. Besides air pollutant levels (ozone and PM2.5), daily temperature and humidity was also selected as covariates in the model and seasonal trends using a natural spline was also adjusted. Medication usage (β-blocker and diabetic medication) was also added to see any modification effects on the association between miRNA levels and air pollutant exposure. Both ozone and PM2.5 were considered as an immediate (lag0), delayed (lag1 to lag4), or cumulative (5 day moving average, 5dMA) linear effect. All outcomes were log transformed before analysis, are reported as percent change from mean of the measured outcome per 1-unit change of air pollutant level (1 unit: ozone 1ppb, PM2.5 1 μg/m3), and statistical significance was set at p < 0.05. Figures were graphed using a GraphPad Prism 8 software.

3. Results

3.1. Subject characteristics and air pollutant measurements

A total of 15 subjects were recruited for the study, but plasma samples for all but one subjects were available for miRNA extraction and data analysis. During the study period (July 2012 to May 2014), 3 out of 14 subjects completed fewer than 4 sessions while the other 11 subjects completed at least 9 sessions. In total, 114 sessions were included for analysis. Most subjects were taking medications: 64.3% beta blockers, 85.7% statins, and 50% diabetic medication (Table 1). Health status of the 14 subjects were also summarized into Table 1. The average BMI of the14 subjects was 31, the average of systolic blood pressure and diastolic blood pressure was 118 and 67, respectively. Among the 14 subjects, 35.7% were previously diagnosed with myocardial infarction, 64.3% had previous hypertension, and 50% were past smokers. Noteworthy, active smokers were excluded from the study at the enrollment.

Table 1.

Subject characteristics.

Characteristics (n=14) Value
Age (years) 62 (43 – 69)
Males 14 (100%)
Race
 Caucasian 11 (78.6%)
 African-American 3 (21.4%)
Current health status
 BMI (kg/m2) 31 (26 – 38)
 Systolic blood pressure 118 (98 – 145)
 Diastolic blood pressure 67 (50 – 86)
Current medication use
 Beta blockers 9 (64.3%)
 Statins 12 (85.7%)
 Diabetic medication 7 (50%)
Past health status
 Previous MI 5 (35.7%)
 Previous hypertension 9 (64.3%)
 Past smokers 7 (50%)
Open in a new tab

Note: Body mass index (BMI), myocardial infarction (MI). Part of the data were adapted from the previous published study (Mirowsky et al., 2017), and the data was edited to reflect the sample included in this study.)

Air pollution measurements corresponding to the study period are shown in Table 2. Due to variations in the level of ozone in any given day, we chose the highest 8-hour ozone concentration for analysis. The 8-hour ozone average across the study period was 36.89 ppb with a range of 5.00 – 86.00 ppb. The 24-hour PM2.5 average (mean: 10.91 μg/m3, range: 1.00 – 28.20 μg/m3) and meteorological measurements for this period were published previously (Mirowsky et al., 2017). Ambient ozone concentrations showed seasonal changes with concentrations being much higher in the summer months. Ambient PM2.5 levels were relatively stable during the study period.

Table 2.

Daily 8-hour average of ozone and 24-hour average of PM2.5 during study period.

Pollutant Mean ± SD Minimum 1st Quartile Median 3rd Quartile Maximum
Ozone (ppb) 36.89 ± 12.18 5.00 28.00 36.00 45.00 86.00
PM2.5 (μg/m3) 10.91 ± 4.51 1.00 7.79 9.93 13.28 28.20
Open in a new tab

SD: standard deviation; ppb: parts per billion; PM: particulate matter.

3.2. Associations between exposure to ambient air pollutants and miRNA expression

A mixed effect model was used to investigate the association between ambient air pollutant levels and EV miRNA expression with covariates of seasonality, temperature, and humidity. As shown in Fig. 1, increases in ambient ozone concentrations (1ppb) were significantly associated with increased miR-150 expression with 1-day lag (1.85%, 95% CI = 0.41%, 3.31%, p = 0.014), 2-day lag (1.65%, 95% CI = 0.53%, 2.79%, p = 0.005), 3-day lag (1.90%, 95% CI = 0.48%, 3.35%, p = 0.010), 4-day lag (1.52%, 95% CI = 0.26%, 2.79%, p = 0.020), and 5-day moving average (3.3%, 95% CI = 1.58%, 5.11%, p < 0.001). Expression of miR-155 in plasma EV was also significantly associated with a 1 ppb increase of ambient ozone concentration at lag 0 (1.72%, 95% CI = 0.04%, 3.40%, p = 0.048). A slight increased expression of miR-21 (2.85%, 95% CI = −0.21%, 5.91%, p = 0.072) was observed with ozone increase at lag 0, although it was not statistically significant. The associations between changes in ambient PM2.5 levels and the expression of any miRNA targets were not statistically significant at any of the time points examined.

Figure 1.

Figure 1.

Open in a new tab

Percent changes of EV miRNA expression levels with ambient ozone and PM2.5 concentrations. Effect estimates (95% CI) were calculated corresponding to changes per 1 ppb of ozone or 1 μg/m3 of PM2.5 and were adjusted for season, temperature, and humidity. 5dMA = 5 day moving average.

3.3. Modified effects by medication usage

We further investigated whether medication usage had any modifying effects on the association between air pollutant levels and miRNA expression. As shown in Table 1, 12 out of 14 subjects used statins leaving a very small sample size for non-users. Thus, we only considered β-blocker (64.3% users) and diabetic medication (50% users) as possible effect modifiers and added them as covariates with interaction with air pollutant in the model. As shown in Fig. 2, β-blocker usage significantly modified the association between ambient ozone increase and miR-150 expression where the parameter estimation showed the associations were only statistically significant among non-users with a 1-day lag (1.96%, 95% CI = 0.36%, 3.59%, p = 0.018), 2-day lag (1.76%, 95% CI = 0.50%, 3.03%, p = 0.007), 3-day lag (2.07%, 95% CI = 0.48%, 3.69%, p = 0.012), 4-day lag (1.58%, 95% CI = 0.11%, 3.07%, p = 0.037), and 5-day moving average (3.54%, 95% CI = 1.61%, 5.51%, p = 0.001). Similarly, significant associations between ambient ozone level changes and miR-150 expression levels were found at 2-day lag (1.83%, 95% CI = 0.52%, 3.16%, p = 0.007) and 3-day lag (2.07%, 95% CI = 0.25%, 3.93%, p = 0.028) among subjects who did not take any diabetic medication, and with a 5-day moving average independent of diabetic medication use (non-users 3.49%, 95% CI = 1.25%, 5.73%, p = 0.003; users 3.17%, 95% CI = 0.76%, 5.65%, p = 0.011) (Fig. 2).

Figure 2.

Figure 2.

Open in a new tab

Percent changes of EV miR-150 expression levels with ambient ozone concentrations adjusted for medication usage. The association between miR-150 expression and ambient ozone exposure were adjusted for (A) β-blocker or (B) for diabetic medication usage. Effect estimates (95% CI) were calculated, corresponding to changes per 1 ppb of ozone and were adjusted for season, temperature, humidity. 5dMA = 5 day moving average.

The associations between increases in ambient ozone levels and the expression of miR-21, miR-126, miR-146, and miR-155 in EV were not significantly modified by either β-blocker or diabetic medication usage, except miR-155 expression was only significantly associated with ozone at 4-day lag among subjects who used diabetic medication (2.80%, 95% CI = 0.06%, 5.62%, p = 0.048) (Fig. S1 and S2). Ambient PM2.5 increase was not significantly associated with the expression of any miRNA targets irrespective of β-blocker usage (Fig. S3). Similarly, the association was not significant between PM2.5 exposure and miRNA expression regardless of diabetic medication usage besides associations between PM2.5 with a 1-day lag and miR-21 expression (−7.29%, 95% CI = −13.85%, −0.23%, p = 0.046), PM2.5 with a 3-day lag and miR-155 expression (−6.19%, 95% CI = −11.75%, −0.27%, p = 0.043) among non-users, and between PM2.5 with a 4-day lag and miR-150 expression (−3.68%, 95% CI = −7.04%, −0.21%, p = 0.040) among users (Fig. S4).

4. Discussion

EV miRNAs are capable of communicating cellular signals between cells or organs (Kao and Papoutsakis, 2019). Genetic regulation by varying levels of miRNA may serve as an important mechanism for inter-organ and inter-tissue communication and ultimately lead to functional changes such as systemic inflammation (Hammond, 2015). Thus, investigating the role of circulating miRNA levels in the association between exposure to air pollutants and cardiovascular diseases will improve our understanding of the effects of air pollution exposure on cardiovascular health. In this study, we found that changes in EV miR-150 and miR-155 in the plasma of CAD patients was significantly associated with exposure to ambient ozone, but not those of PM2.5.

Only a few studies investigated whether exposure to ozone can lead to changes in miRNA expression, mostly focusing on inflammation and immune responses in the respiratory system. A controlled human exposure study found that exposure to 0.4 ppm ozone for 2 hours significantly increased the expression of 10 miRNAs including miR-132, miR-143, miR-145, miR-199a in induced sputum samples from healthy adult volunteers (Fry et al., 2014). Further pathway analysis showed these miRNAs were highly associated with inflammation and immune-related diseases (Fry et al., 2014). Another animal study showed that the conducting airway epithelium of neonates might be a target of immunomodulation of ozone, and reported that episodic ozone exposure of juvenile rhesus macaque monkeys resulted in immune responses that might be regulated by differentially expressed miRNAs such as miR-149 (Clay et al., 2014). Exposure to 2 ppm of ozone for 3 hours altered expression levels of miRNAs in lung tissues which were associated with IL-6 inflammatory pathway in mice (Fuentes et al., 2018). One study investigating the role of miRNA in the cardiovascular effects of ozone exposure in male Wistar Kyoto rats found that the miRNA profiles in the serum showed no significant changes after 0.8 ppm ozone exposure (Snow et al., 2018). These human and animal studies have all employed a short-term exposure to much higher ozone doses than the ambient level that we presented in our current study. Though not likely to apply to “real-life” exposures, most of these studies have found significant changes of miRNAs that are associated with inflammatory responses. Yet results from our clinical study would be the first to show that ambient ozone exposure can lead to changes in circulating miRNA levels with a focus on cardiovascular effects.

The significant association between miR-150 expression and ambient ozone exposure for up to 4 days prior suggest that this biomarker is very sensitive to ozone exposure and may serve as an important link to ozone induced cardiovascular effects. In terms of other pollutants, one study found that long-term (1 year) exposure to ambient PM2.5 was significantly associated with miR-150 expression in plasma among older adults (Rodosthenous et al., 2016). Increased miR-150 levels have shown protective effects against cardiovascular disease severity. Clinical studies showed that miR-150 levels were significantly higher in the early stages of acute myocardial infarction, while lower levels of miR-150 were associated with increased mortality due to acute ischemic stroke (Li et al., 2019; Scherrer et al., 2017). Increased expression of miR-150 was often found among CAD patients and was associated with protective effects against ischemic injury by inhibiting monocyte influx and affecting ventricular remodeling after myocardial infraction or cardiac hypertrophy (Qiu et al., 2019). Studies have also found miR-150 can restore endothelial function and reduce vascular remodeling through Pentraxin-related protein 3 and the NFκB pathway (Luo et al., 2018). The association between miR-150 expression and ambient ozone was even more prominent among subjects who were not taking diabetic medications or β-blockers. Similarly, miR-150 expression was downregulated by medication usage (e.g. ACE inhibitors, β-blockers, statins, aspirin, etc) among coronary heart disease patients (Li et al., 2019). These findings suggest that use of such medications can lower miRNA levels and further maintain homeostasis for miRNA-mediated functional changes. While how medications, such as β-blocker, regulate circulating miRNA levels remain unclear, mechanisms of how these medications treat cardiovascular disease can point to the direction of future research. For example, nebivolol, a type of β-blocker, can treat cardiac remodeling through activating the beta-2 adrenergic receptor (AR) pathway that involve proteins including FOXO1, BCL2, PIK3R1, and TGFB3, which are also regulating targets of miR-27a and miR-29a (Tran Quang et al., 2009; Yang et al., 2014). We could speculate that the direct protective effects from the β-blocker on the cardiac remodeling were also reflected on the interaction between the proteins on the beta-2 AR pathway and the varying levels of miRNAs.

The positive association between miR-155 expression and ambient ozone exposure was only significant on day 0, indicating a short-term effect of ozone on circulating miR-155 levels. Increased levels of miR-155 expression are believed to be protective in terms of relieving chronic inflammation linked to atherosclerosis-associated foam cell formation (Li et al., 2016). A few studies have investigated the role of miR-155 in air pollutants induced health effects. One study showed serum levels of miR-155 were significantly linked to indoor PM2.5 and formaldehyde levels among children with asthma (Liu et al., 2019). Elevated miR-155 expression has also been related to exposure to environmental toxicants such as polycyclic aromatic hydrocarbons in health adults (Ruiz-Vera et al., 2019). An acute increase in miR-155 in response to ambient ozone exposure could elicit some protective regulatory mechanisms. In a mechanistic study, human bronchial epithelial cells exposed to PM2.5 showed up-regulation of miR-155 in a dose-dependent manner, and the change was showed to be mediated by epigenetic modulation of the SOCSC1/STAT3 signaling pathway (Xiao et al., 2019b). On the other hand, several studies showed reduced circulating levels of miR-155 among CAD patients (Faccini et al., 2017; Jia et al., 2017; Zhao et al., 2018), indicating a possible overwhelmed miR-155 regulatory mechanism in prolonged disease states. It should be noted that as all subjects in our study were CAD patients, and that it is therefore possible that they may already have a relatively low miR-155 expression in their circulation.

Of the five miRNA targets in this study, three were not significantly associated with either ambient PM2.5 or ozone exposure. However, these three targets have the ability to regulate biological responses in the cardiovascular system. Circulating miR-21 was associated with disease onset and progression in cardiovascular diseases, especially in cell proliferation and apoptosis. Upregulation of miR-21 was protective against oxidative stress induced apoptosis of cardiomyocytes through downregulating the expression of programmed cell death 4 (PDCD4) (Xiao et al., 2016), and also against diabetic cardiomyopathy by inhibiting gelsolin expression (Dai et al., 2018). Ablation of miR-21 expression in vitro exacerbated aldosterone-mediated cardiac injury, remodeling, and dysfunction (Syed et al., 2018). Increased levels of miR-126 in the circulation have been proven to be novel biomarkers for diagnosis for acute myocardial infarction (Xue et al., 2019), but have also been correlated with lower disease risk and severity, as well as reduced inflammatory cytokine levels in acute ischemic stroke patients (Jin and Xing, 2018). Wang et. al. found that increased miR-126 expression can attenuate oxidative stress and cell apoptosis caused by ischemia and reperfusion through targeting ERBB receptor feedback inhibitor 1 (Wang et al., 2019). Similarly, among cardiovascular disease patients, miR-146 expression levels were significantly elevated in the circulation, cells and tissues in the cardiovascular system (Paterson and Kriegel, 2017). Elevated miR-146 can negatively regulate pro-inflammatory pathways such as TLR4 (toll like receptor 4) -dependent mechanisms (Xiao et al., 2019a) and is also involved in control of atherosclerosis induced by exposure to trimethylamine-N-oxide (TMAO) (Coffey et al., 2019). In this study, there were no significant changes in the expression of miR-21, miR-126, or miR-146 associated with ambient air pollutant levels. A study by Nariman-Saleh-Fam et al (Nariman-Saleh-Fam et al., 2019) suggests that subjects at different stages of coronary stenosis have different miRNA expression profiles and extent of each miRNA target change can also vary. It is possible that these miRNA targets have already reached a high level due to the subject’s CAD stages and were thus less sensitive to ambient air pollution in this study.

Though not observed in this study, a few others have reported significant association between miRNA changes in the circulation and PM2.5-induced cardiovascular effects. Among an older population, researchers found associations between long-term exposure to ambient PM2.5 and changed levels of miRNA targets such as miR-126, miR-19b, miR-150, miR-191, and let-7a (Rodosthenous et al., 2016). Similar to our findings, one study focused on miRNA profiles with short-term exposure to traffic-related air pollutants (NO, NO2, CO, and CO2) did not find any significant association between exposure to PM2.5 or PM10 and miRNA changes in the circulation (Krauskopf et al., 2019). The discrepancy between our results and the aforementioned studies might come from following aspects. First, subjects from our study were all existing CAD patients and their relatively advanced disease may have already significantly changed levels of the targeted miRNAs, meaning that exposure to ambient PM2.5 changes may not have induced further changes. Second, our targeted approach focusing on five miRNA targets may not have included those most sensitive to PM2.5 changes. Third, the fact that ambient PM2.5 levels across the study remained relatively low with minimal variance, there may not have been enough power to detect a relationship between ambient levels of PM2.5 and changes in circulating miRNA levels.

In all, we believe, increase levels of miR-150 and miR-155 may not only become markers of exposure to ambient levels of ozone, but also serve as point of research for the mechanisms of ozone-induced cardiovascular effects. A similar study has already investigated the modifying mechanism, which Rodosthenous and colleagues had showed that miRNA expression changes as a result of DNA methylation regulation, can modify the association between PM2.5 and systolic blood pressure (Rodosthenous et al., 2018). To validate it in our case, future research will examine the modifying effects of miR-150 and miR-155 between ozone exposure and cardiovascular endpoints. Although this panel study had repeated measurements for each subject, the sample size was relatively small with only 14 subjects and 114 data points. A larger sample size would provide greater statistical power to detect associations. In addition, outdoor air pollution measurements used in the study do not account for indoor infiltration (i.e., attenuation, passive smoking in the household) of ambient PM2.5 and ozone that can vary from house-to-house, day-to-day, and time spent in different indoor and outdoor microenvironments (Breen et al., 2018; Breen, 2020; Breen et al., 2015). Multiple mechanistic pathways are involved in air pollution-induced cardiovascular health effects (e.g. direct damage, systemic inflammation, EV miRNA changes, etc). Thus, future studies should also take into considerations these factors. Nevertheless, our findings are the first to report an association between ambient ozone exposure and circulating miRNA levels and shed light on a research direction which focuses on the miRNA-mediated cardiovascular effects caused by human exposure to ambient air pollution.

5. Conclusions

In this study, we found that increased expression levels of EV miR-150 at lag1–4 days and miR-155 at lag 0 were significantly associated with exposure to ambient ozone, not PM2.5. The association between miR-150 expression and ozone exposure was more prominent among non-medicated subjects. These preliminary findings will contribute to understanding the mechanisms of air pollutant induced cardiovascular health effects and warrant for more future researches.

Supplementary Material

Supplement1

Acknowledgements:

The authors thank Dr. James Samet and Dr. Michael Breen for their review and suggestions.

Funding: The U.S. Environmental Protection Agency Intramural Research Program supported this research.

Footnotes

Competing interests: The authors declare that they have no actual or competing interests.

Publisher's Disclaimer: Disclaimer: The research described in this article has been reviewed by the Center for Public Health and Environmental Assessment, U.S. EPA, and approved for publication. The contents of this article should not be construed to represent Agency policy nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

References

  1. An Z, Jin Y, Li J, Li W, Wu W, 2018. Impact of Particulate Air Pollution on Cardiovascular Health. Curr Allergy Asthma Rep 18, 15. [DOI] [PubMed] [Google Scholar]
  2. Breen M, Xu Y, Schneider A, Williams R, Devlin R, 2018. Modeling individual exposures to ambient PM2.5 in the diabetes and the environment panel study (DEPS). Sci Total Environ 626, 807–816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breen MC, S.Y.; Breen M; Xu Y; Isakov V; Arunachalam S; Carraway MS; Devlin R, 2020. Fine-Scale Modeling of Individual Exposures to Ambient PM2.5, EC, NOx, and CO for the Coronary Artery Disease and Environmental Exposure (CADEE) Study. Atmosphere 11, 65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breen MS, Long TC, Schultz BD, Williams RW, Richmond-Bryant J, Breen M, Langstaff JE, Devlin RB, Schneider A, Burke JM, Batterman SA, Meng QY, 2015. Air Pollution Exposure Model for Individuals (EMI) in Health Studies: Evaluation for Ambient PM2.5 in Central North Carolina. Environ Sci Technol 49, 14184–14194. [DOI] [PubMed] [Google Scholar]
  5. Breitner S, Schneider A, Devlin RB, Ward-Caviness CK, Diaz-Sanchez D, Neas LM, Cascio WE, Peters A, Hauser ER, Shah SH, Kraus WE, 2016. Associations among plasma metabolite levels and short-term exposure to PM2.5 and ozone in a cardiac catheterization cohort. Environ Int 97, 76–84. [DOI] [PubMed] [Google Scholar]
  6. Bronze-da-Rocha E, 2014. MicroRNAs expression profiles in cardiovascular diseases. Biomed Res Int 2014, 985408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brook RD, Rajagopalan S, Pope CA 3rd, Brook JR, Bhatnagar A, Diez-Roux AV, Holguin F, Hong Y, Luepker RV, Mittleman MA, Peters A, Siscovick D, Smith SC Jr., Whitsel L, Kaufman JD, American Heart Association Council on, E., Prevention, C.o.t.K.i.C.D., Council on Nutrition, P.A., Metabolism, 2010. Particulate matter air pollution and cardiovascular disease: An update to the scientific statement from the American Heart Association. Circulation 121, 2331–2378. [DOI] [PubMed] [Google Scholar]
  8. Cao RY, Li Q, Miao Y, Zhang Y, Yuan W, Fan L, Liu G, Mi Q, Yang J, 2016. The Emerging Role of MicroRNA-155 in Cardiovascular Diseases. Biomed Res Int 2016, 9869208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng Y, Zhang C, 2010. MicroRNA-21 in cardiovascular disease. J Cardiovasc Transl Res 3, 251–255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chuang KJ, Chan CC, Su TC, Lee CT, Tang CS, 2007. The effect of urban air pollution on inflammation, oxidative stress, coagulation, and autonomic dysfunction in young adults. Am J Respir Crit Care Med 176, 370–376. [DOI] [PubMed] [Google Scholar]
  11. Clay CC, Maniar-Hew K, Gerriets JE, Wang TT, Postlethwait EM, Evans MJ, Fontaine JH, Miller LA, 2014. Early life ozone exposure results in dysregulated innate immune function and altered microRNA expression in airway epithelium. PLoS One 9, e90401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coffey AR, Kanke M, Smallwood TL, Albright J, Pitman W, Gharaibeh RZ, Hua K, Gertz E, Biddinger SB, Temel RE, Pomp D, Sethupathy P, Bennett BJ, 2019. microRNA-146a-5p association with the cardiometabolic disease risk factor TMAO. Physiol Genomics 51, 59–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Collaborators, G.B.D.R.F., 2018. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1923–1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dai B, Li H, Fan J, Zhao Y, Yin Z, Nie X, Wang DW, Chen C, 2018. MiR-21 protected against diabetic cardiomyopathy induced diastolic dysfunction by targeting gelsolin. Cardiovasc Diabetol 17, 123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Day DB, Xiang J, Mo J, Li F, Chung M, Gong J, Weschler CJ, Ohman-Strickland PA, Sundell J, Weng W, Zhang Y, Zhang JJ, 2017. Association of Ozone Exposure With Cardiorespiratory Pathophysiologic Mechanisms in Healthy Adults. JAMA Intern Med 177, 1344–1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Faccini J, Ruidavets JB, Cordelier P, Martins F, Maoret JJ, Bongard V, Ferrieres J, Roncalli J, Elbaz M, Vindis C, 2017. Circulating miR-155, miR-145 and let-7c as diagnostic biomarkers of the coronary artery disease. Sci Rep 7, 42916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fernandez-Hernando C, Suarez Y, 2018. MicroRNAs in endothelial cell homeostasis and vascular disease. Curr Opin Hematol 25, 227–236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fry RC, Rager JE, Bauer R, Sebastian E, Peden DB, Jaspers I, Alexis NE, 2014. Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects. Am J Physiol Lung Cell Mol Physiol 306, L1129–1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fuentes N, Roy A, Mishra V, Cabello N, Silveyra P, 2018. Sex-specific microRNA expression networks in an acute mouse model of ozone-induced lung inflammation. Biol Sex Differ 9, 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Furuyama A, Kanno S, Kobayashi T, Hirano S, 2009. Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes. Arch Toxicol 83, 429–437. [DOI] [PubMed] [Google Scholar]
  21. Ghio AJ, Kim C, Devlin RB, 2000. Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. Am J Respir Crit Care Med 162, 981–988. [DOI] [PubMed] [Google Scholar]
  22. Hammond SM, 2015. An overview of microRNAs. Adv Drug Deliv Rev 87, 3–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heron M, 2018. Deaths: Leading Causes for 2016. Natl Vital Stat Rep 67, 1–77. [PubMed] [Google Scholar]
  24. Jia QW, Chen ZH, Ding XQ, Liu JY, Ge PC, An FH, Li LH, Wang LS, Ma WZ, Yang ZJ, Jia EZ, 2017. Predictive Effects of Circulating miR-221, miR-130a and miR-155 for Coronary Heart Disease: A Multi-Ethnic Study in China. Cell Physiol Biochem 42, 808–823. [DOI] [PubMed] [Google Scholar]
  25. Jin F, Xing J, 2018. Circulating miR-126 and miR-130a levels correlate with lower disease risk, disease severity, and reduced inflammatory cytokine levels in acute ischemic stroke patients. Neurol Sci 39, 1757–1765. [DOI] [PubMed] [Google Scholar]
  26. Kao CY, Papoutsakis ET, 2019. Extracellular vesicles: exosomes, microparticles, their parts, and their targets to enable their biomanufacturing and clinical applications. Curr Opin Biotechnol 60, 89–98. [DOI] [PubMed] [Google Scholar]
  27. Krauskopf J, van Veldhoven K, Chadeau-Hyam M, Vermeulen R, Carrasco-Turigas G, Nieuwenhuijsen M, Vineis P, de Kok TM, Kleinjans JC, 2019. Short-term exposure to traffic-related air pollution reveals a compound-specific circulating miRNA profile indicating multiple disease risks. Environ Int 128, 193–200. [DOI] [PubMed] [Google Scholar]
  28. Lawal AO, 2017. Air particulate matter induced oxidative stress and inflammation in cardiovascular disease and atherosclerosis: The role of Nrf2 and AhR-mediated pathways. Toxicol Lett 270, 88–95. [DOI] [PubMed] [Google Scholar]
  29. Li H, Zhang P, Li F, Yuan G, Wang X, Zhang A, Li F, 2019. Plasma miR-22–5p, miR-132–5p, and miR-150–3p Are Associated with Acute Myocardial Infarction. Biomed Res Int 2019, 5012648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li X, Kong D, Chen H, Liu S, Hu H, Wu T, Wang J, Chen W, Ning Y, Li Y, Lu Z, 2016. miR-155 acts as an anti-inflammatory factor in atherosclerosis-associated foam cell formation by repressing calcium-regulated heat stable protein 1. Sci Rep 6, 21789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu Q, Wang W, Jing W, 2019. Indoor air pollution aggravates asthma in Chinese children and induces the changes in serum level of miR-155. Int J Environ Health Res 29, 22–30. [DOI] [PubMed] [Google Scholar]
  32. Louwies T, Vuegen C, Panis LI, Cox B, Vrijens K, Nawrot TS, De Boever P, 2016. miRNA expression profiles and retinal blood vessel calibers are associated with short-term particulate matter air pollution exposure. Environ Res 147, 24–31. [DOI] [PubMed] [Google Scholar]
  33. Luo XY, Zhu XQ, Li Y, Wang XB, Yin W, Ge YS, Ji WM, 2018. MicroRNA-150 restores endothelial cell function and attenuates vascular remodeling by targeting PTX3 through the NF-kappaB signaling pathway in mice with acute coronary syndrome. Cell Biol Int 42, 1170–1181. [DOI] [PubMed] [Google Scholar]
  34. Medina-Ramon M, Schwartz J, 2008. Who is more vulnerable to die from ozone air pollution? Epidemiology 19, 672–679. [DOI] [PubMed] [Google Scholar]
  35. Mirowsky JE, Carraway MS, Dhingra R, Tong H, Neas L, Diaz-Sanchez D, Cascio W, Case M, Crooks J, Hauser ER, Elaine Dowdy Z, Kraus WE, Devlin RB, 2017. Ozone exposure is associated with acute changes in inflammation, fibrinolysis, and endothelial cell function in coronary artery disease patients. Environ Health 16, 126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, de Ferranti S, Despres JP, Fullerton HJ, Howard VJ, Huffman MD, Judd SE, Kissela BM, Lackland DT, Lichtman JH, Lisabeth LD, Liu S, Mackey RH, Matchar DB, McGuire DK, Mohler ER 3rd, Moy CS, Muntner P, Mussolino ME, Nasir K, Neumar RW, Nichol G, Palaniappan L, Pandey DK, Reeves MJ, Rodriguez CJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Willey JZ, Woo D, Yeh RW, Turner MB, American Heart Association Statistics, C., Stroke Statistics, S., 2015. Heart disease and stroke statistics–-2015 update: a report from the American Heart Association. Circulation 131, e29–322. [DOI] [PubMed] [Google Scholar]
  37. Nariman-Saleh-Fam Z, Vahed SZ, Aghaee-Bakhtiari SH, Daraei A, Saadatian Z, Kafil HS, Yousefi B, Eyvazi S, Khaheshi I, Parsa SA, Moravej A, Mousavi N, Bastami M, Mansoori Y, 2019. Expression pattern of miR-21, miR-25 and PTEN in peripheral blood mononuclear cells of patients with significant or insignificant coronary stenosis. Gene 698, 170–178. [DOI] [PubMed] [Google Scholar]
  38. Nuvolone D, Balzi D, Pepe P, Chini M, Scala D, Giovannini F, Cipriani F, Barchielli A, 2013. Ozone short-term exposure and acute coronary events: a multicities study in Tuscany (Italy). Environ Res 126, 17–23. [DOI] [PubMed] [Google Scholar]
  39. Paterson MR, Kriegel AJ, 2017. MiR-146a/b: a family with shared seeds and different roots. Physiol Genomics 49, 243–252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pereira-da-Silva T, Coutinho Cruz M, Carrusca C, Cruz Ferreira R, Napoleao P, Mota Carmo M, 2018. Circulating microRNA profiles in different arterial territories of stable atherosclerotic disease: a systematic review. Am J Cardiovasc Dis 8, 1–13. [PMC free article] [PubMed] [Google Scholar]
  41. Qiu M, Ma J, Zhang J, Guo X, Liu Q, Yang Z, 2019. MicroRNA-150 deficiency accelerates intimal hyperplasia by acting as a novel regulator of macrophage polarization. Life Sci, 116985. [DOI] [PubMed] [Google Scholar]
  42. Rodosthenous RS, Coull BA, Lu Q, Vokonas PS, Schwartz JD, Baccarelli AA, 2016. Ambient particulate matter and microRNAs in extracellular vesicles: a pilot study of older individuals. Part Fibre Toxicol 13, 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rodosthenous RS, Kloog I, Colicino E, Zhong J, Herrera LA, Vokonas P, Schwartz J, Baccarelli AA, Prada D, 2018. Extracellular vesicle-enriched microRNAs interact in the association between long-term particulate matter and blood pressure in elderly men. Environ Res 167, 640–649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ruiz-Vera T, Ochoa-Martinez AC, Pruneda-Alvarez LG, Dominguez-Cortinas G, Perez-Maldonado IN, 2019. Expression levels of circulating microRNAs-126, −155, and −145 in Mexican women exposed to polycyclic aromatic hydrocarbons through biomass fuel use. Environ Mol Mutagen 60, 546–558. [DOI] [PubMed] [Google Scholar]
  45. Salvi S, Blomberg A, Rudell B, Kelly F, Sandstrom T, Holgate ST, Frew A, 1999. Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am J Respir Crit Care Med 159, 702–709. [DOI] [PubMed] [Google Scholar]
  46. Scherrer N, Fays F, Mueller B, Luft A, Fluri F, Christ-Crain M, Devaux Y, Katan M, 2017. MicroRNA 150–5p Improves Risk Classification for Mortality within 90 Days after Acute Ischemic Stroke. J Stroke 19, 323–332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmittgen TD, Livak KJ, 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101–1108. [DOI] [PubMed] [Google Scholar]
  48. Snow SJ, Cheng WY, Henriquez A, Hodge M, Bass V, Nelson GM, Carswell G, Richards JE, Schladweiler MC, Ledbetter AD, Chorley B, Gowdy KM, Tong H, Kodavanti UP, 2018. Ozone-Induced Vascular Contractility and Pulmonary Injury Are Differentially Impacted by Diets Enriched With Coconut Oil, Fish Oil, and Olive Oil. Toxicol Sci 163, 57–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Syed M, Ball JP, Mathis KW, Hall ME, Ryan MJ, Rothenberg ME, Yanes Cardozo LL, Romero DG, 2018. MicroRNA-21 ablation exacerbates aldosterone-mediated cardiac injury, remodeling, and dysfunction. Am J Physiol Endocrinol Metab 315, E1154–E1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tran Quang T, Rozec B, Audigane L, Gauthier C, 2009. Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta. Br J Pharmacol 156, 601–608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van Eeden SF, Tan WC, Suwa T, Mukae H, Terashima T, Fujii T, Qui D, Vincent R, Hogg JC, 2001. Cytokines involved in the systemic inflammatory response induced by exposure to particulate matter air pollutants (PM(10)). Am J Respir Crit Care Med 164, 826–830. [DOI] [PubMed] [Google Scholar]
  52. Wang W, Zheng Y, Wang M, Yan M, Jiang J, Li Z, 2019. Exosomes derived miR-126 attenuates oxidative stress and apoptosis from ischemia and reperfusion injury by targeting ERRFI1. Gene 690, 75–80. [DOI] [PubMed] [Google Scholar]
  53. WHO, 2018. The top 10 causes of death.
  54. Xiao J, Pan Y, Li XH, Yang XY, Feng YL, Tan HH, Jiang L, Feng J, Yu XY, 2016. Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4. Cell Death Dis 7, e2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xiao Q, Zhu X, Yang S, Wang J, Yin R, Song J, Ma A, Pan X, 2019a. LPS induces CXCL16 expression in HUVECs through the miR-146a-mediated TLR4 pathway. Int Immunopharmacol 69, 143–149. [DOI] [PubMed] [Google Scholar]
  56. Xiao T, Ling M, Xu H, Luo F, Xue J, Chen C, Bai J, Zhang Q, Wang Y, Bian Q, Liu Q, 2019b. NF-kappaB-regulation of miR-155, via SOCS1/STAT3, is involved in the PM2.5-accelerated cell cycle and proliferation of human bronchial epithelial cells. Toxicol Appl Pharmacol 377, 114616. [DOI] [PubMed] [Google Scholar]
  57. Xue S, Liu D, Zhu W, Su Z, Zhang L, Zhou C, Li P, 2019. Circulating MiR-17–5p, MiR-126–5p and MiR-145–3p Are Novel Biomarkers for Diagnosis of Acute Myocardial Infarction. Front Physiol 10, 123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yang J, Liu Y, Fan X, Li Z, Cheng Y, 2014. A pathway and network review on beta-adrenoceptor signaling and beta blockers in cardiac remodeling. Heart Fail Rev 19, 799–814. [DOI] [PubMed] [Google Scholar]
  59. Zhao D, Zhao J, Sun J, Su Y, Jian J, Ye H, Lin J, Yang Z, Feng J, Wang Z, 2018. The expression level of miR-155 in plasma and peripheral blood mononuclear cells in coronary artery disease patients and the associations of these levels with the apoptosis rate of peripheral blood mononuclear cells. Exp Ther Med 16, 4373–4378. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement1
NIHMS1598448-supplement-Supplement1.docx (219KB, docx)

RESOURCES