In this study, we investigated the hypothesis that the inhalation of DE under both acute (1-week, 1-month) and chronic (3-month, 6-month) conditions would exacerbate the progression of cardiac hypertrophy and failure in two models of cardiac hypertrophy in mice. Although there is strong evidence that exposure to traffic related air pollution results in increased left ventricular mass in humans [7, 8], and results in increased hypertrophy as well as systolic and diastolic dysfunction in mice , we did not observe any effect of DE exposure alone on the hypertrophic or contractile responses in two mouse models of cardiac hypertrophy.
We did observe a paradoxical effect of DE exposure on heart rate. In epidemiological studies, the general consensus is that HR increases while measures of HRV decrease in humans exposed to PM . However, we see the opposite effect in this study. The reason for this may be due to differences between rodents and humans. Other studies have seen similar results with exposure to CAPs  and carbon black . It is possible that although the autonomic nervous system (ANS) is affected by the DE exposure, afferent ANS pathways differ in mice from those in humans.
There are some caveats to our study. Although we do not observe any effects of DE exposure on hypertrophy or heart function at the organ level, it is possible that activation of pathways at the molecular level has occurred. Even if these changes are occurring, however, the lack of response at the organ level indicates that these hypertrophy models and this exposure system may be limited in their utility for studying cardiovascular pathogenetic mechanisms induced by DE exposure. Another caveat is that our echocardiographic assessment was done from 1–7 days after diesel exposure was terminated, and we cannot rule out short lived effects on cardiac function that disappear rapidly after exposure is terminated. Others have found, however, that CAP exposure can lead to significant effects on cardiac function two weeks after the exposure is terminated . Using the same exposure facility and conditions described here, we have also found that exposure to DE exhaust in utero and early in life can predispose to hypertrophy in adults, long after the exposure is terminated (Weldy CS et al., manuscript submitted). Another caveat to our study is that we did not measure blood pressure in the animals that were treated with Angiotensin II, and there is a possibility that the minipumps failed to deliver appropriate amounts. Our observation of Ang II induced increases in VW/BW and LV WT in Figure 1 is strongly indicative that Ang II was infused appropriately. Angiotensin II is also known to promote cardiac hypertrophy at subpressor doses, obviating the need for blood pressure monitoring . Still another potential caveat is that we did not assess for either pulmonary or systemic inflammation in our mice. We and others have shown that similar exposures using our facility result in low level neutrophilic airway inflammation  and systemic lipid peroxidation . We expect similar results in the mice used in this study.
Controlled exposures to DE in both human and animal studies have been used routinely to model ambient PM2.5 exposure. The benefits of using DE have been the relative ease of reproducible and well-defined exposures as well as the ability to compare biological effects of DE across institutions. In addition, in many regions, the majority of ambient PM2.5 is derived from diesel engines, thus DE exposures are largely mimicking ambient exposures to urban and traffic related particulate air pollution. Studies utilizing collected diesel exhaust particulate (DEP) in both in vitro and in vivo exposures have demonstrated their robust proinflammatory effects and their strong potential to influence systemic vascular function [26–29], but there is strong evidence to suggest that the inflammatory and toxic potential of these particles is influenced by additional particulate constituents, such as endotoxin and metals.
The disadvantage of using DE is that it is a relatively ‘simple’ exposure model, in that the PM2.5 resulting from DE is mostly carbonaceous in nature, containing polycyclic aromatic hydrocarbons and redox active quinones, but lacking many other contaminants important to particulate matter toxicity. In the work reported in Ying et al.  and Wold et al. , they report their observed effects on cardiac hypertrophy and failure after exposure to CAPs from the Columbus, Ohio region. In both of these studies, exposures were done using ambient particulate material that was concentrated directly from the atmosphere, resulting in an exposure that consists of PM2.5 from traffic related sources such as diesel exhaust, but also from jet fuel (as an airport is near the exposure site), agriculture (which may result in endotoxin exposure), as well as coal fired power plants and other industrial sources of PM2.5. The advantage of these CAPs studies is that the PM2.5 exposures closely match the human exposures in the population. The main disadvantage is that these exposures are difficult to compare across institutions as PM2.5 composition will vary widely across regions and proximity to sources, and the PM2.5 composition during the exposure may vary from day-to-day depending on the magnitude of PM2.5 emissions near the exposure site.
The results from the studies reported here suggest that a DE exposure in C57BL/6 J mice up to 6 months in length has no effect on the cardiac hypertrophic or functional responses in Ang II infusion or TAC models of heart failure. That we do not observe any effect in these studies whereas others have reported effects using CAPs suggests that either 1) 6 months exposure to DE is not enough time to see any effect, or 2) DE exposures are lacking PM constituents found in CAPs that provide for their unique cardiotoxicity.
It is possible that a 6 month exposure to DE is not enough time to elicit the hypertrophic or functional effects on the mouse heart. In the report published by Wold et al. , their CAP exposure was for 9 months, and this exposure resulted in increased expression of hypertrophic markers, decreased fractional shortening, diastolic dysfunction and decreased contractile reserve, consistent with heart failure. In our exposures, diesel exhaust concentration is dynamically regulated to 300 μg/m3, 6 hrs a day, 5 days a week. Since mice are exposed to FA for 18 hrs a day, and during weekends, our total time weighted average exposure for the 6 month period is expected to be ~53 μg/m3/hr. We calculate cumulative exposure, using concentration x time, to be 53 μg/hr × 6 months × 30 days/month × 24 hrs/day = 228960 μg/m3. In the study by Wold et al. , they calculated a time weighted average exposure to be 15 μg/m3/hr. The calculated cumulative exposure over 9 months is 15 μg/m3 × 9 months × 30 days/month × 24 hrs/day = 97200 μg /m3. In our assessment, our cumulative exposure to PM2.5 in 6 months is 2.36 times greater than their exposure in 9 months. This suggests to us that our inability to find any significant effect on cardiac hypertrophy or function with our 6-month DE exposure must be due to either 1) constituents of CAPs within the Wold et al. study that elicit greater cardiotoxicity than PM2.5 from DE, or 2) duration of PM2.5 exposure is more important than cumulative exposure. Although, from this assessment, we cannot rule out the possibility that the duration of exposure is the critical component to observing cardiotoxicity, we believe this is unlikely, as our 2.36 times greater cumulative PM2.5 exposure would be expected to elicit a more rapid acceleration of cardiac hypertrophy and failure. In addition, although Wold et al.  had a longer exposure time than the studies reported here, Ying et al.  exposed mice to CAPs for only 3 months, and then assessed their susceptibility to Ang II-induced cardiac hypertrophy. They observed the 3-month CAPs exposure to promote cardiac remodeling induced by a two-week infusion of Ang II through a Rho kinase dependent mechanism. The report by Ying et al.  suggests that a 3-month exposure to CAPs is sufficient to observe these adverse effects resulting from Ang II infusion. In this study, their cumulative PM2.5 exposure was 15 μg/m3/hr × 3 months × 30 days/month × 24 hrs/day = 32400 μg/m3. Our study has a cumulative exposure that is 7 times greater than their study, further suggesting that our lack of finding a significant effect is likely due to diesel exhaust particulate lacking PM constituents that elicit unique responses to the myocardium.
There is a growing belief that particular constituents of ambient PM2.5 likely have important modifications to their toxicity within human populations. In a recent report by Wu et al. , acute PM2.5 exposures in a Chinese population were associated with increases in systolic as well as diastolic blood pressure, and these increases in BP were found to be positively modified by PM2.5 constituents such as carbonaceous fractions, ions, and metals. In addition, a recent report by Kim et al.  observed PM2.5 constituents such as sulfate and nitrate to alter their observed temporal relationships between increases in PM2.5 and hospital admissions. The potential for PM constituents to alter their cytotoxic and proinflammatory effects have been investigated in vitro, where the presence of metals such as arsenic, zinc, chromium, copper, manganese, and iron have been found to be positively associated with deleterious effects [32–35]. As the cytotoxic and proinflammatory effects of PM2.5 have been suggested to be largely mediated by the generation of reactive oxygen species and subsequent oxidative stress, it is likely the case that metal constituents that can participate in redox cycling and Fenton chemistry are capable of enhancing PM2.5 toxicity. As we do not observe a chronic DE exposure (which is largely carbonaceous, containing low metal content) to elicit changes in cardiac hypertrophy or function, it is possible that metal constituents within CAPs are likely enhancing PM2.5 effects on pulmonary and systemic inflammation, potentially driving these changes in ventricular remodeling. These observations may suggest that a certain ‘threshold’ of inflammation and oxidative stress is required to be exceeded before changes in cardiac size or function will be observed. In support of this hypothesis, it has been recently reported that rapid, high dose exposures of collected DEP to rats via intranasal nebulization over a 5-week period can elicit cardiac dysfunction and remodeling , suggesting that DEP may be able to elicit these effects, but it may require extremely high doses, rapid administration, and lengthy exposure times that exceed what would be required when particle constituents within CAPs are included.