We have here demonstrated that, in the murine apolipoprotein E deficiency model, instillation of DEP increased lesion size, produced more lesions per vessel and generated more buried fibrous caps. The proatherosclerotic effect was concomitant with pulmonary inflammation and systemic oxidative stress. We conclude that the particulate components of DE accelerate plaque development and are associated with a more complex plaque phenotype. The processes involved may contribute to the association between exposure to urban air pollution, atherosclerosis and acute myocardial infarction in man.
The influence of DEP was assessed using a recognised model of atherosclerosis at a period of extensive plaque expansion and remodelling [25, 26]. Serial sections of artery were used to provide a surrogate marker of plaque volume and a sensitive approach to quantifying changes in plaque burden. We found that four weeks of repeated DEP exposure caused an almost doubling of plaque size from 32% to 59%. The 4-week exposure to DEP was consistent with pre-clinical investigations using concentrated ambient particles (CAPs) [8, 9, 27–30], whole DE [11, 19] and, to a lesser extent, non-carbon nanoparticles . These previous studies have been performed in several different animal models (ApoE−/− mice, low density lipoprotein receptor Apo E double knockout mice and Watanabe heritable hyperlipidaemic rabbits) using either inhalation [8, 10, 11, 19, 30] or instillation [27, 29, 31]. The present study is the first to assess whether direct exposure to DEP increases atherosclerosis.
The marked increases in plaque size with DEP instillation are striking given the short duration of the exposure period. In the present study, long-term exposure has been emulated by administration of higher doses of particles over a shorter length of time. The instillation technique has been well validated [32–36] and the dose employed is similar to that of other studies using repeated instillations [37, 38], and below those commonly used for single instillation exposures. While we cannot rule out that a proportion of the particulate administered deposits in the upper airways, studies have shown that the aspiration instillation technique to provide excellent delivery and dispersity of particulate throughout the alveoli [39, 40]. The MPPD particle deposition model  estimates that the average 24-h instilled dose used here (assuming all DEP reaches the alveoli) is approximately 40-fold higher than the comparable alveolar deposition from a 24-h inhalation of 100 μg/m3 in man (adjusted for mice). It is important to highlight that these calculations are based on inhalation of particles with a primary particle diameter of that of diesel exhaust (~60 nm), whereas, urban levels of air pollution will contain a wide range of particulate sizes of which only a lower proportion of the larger particulates will penetrate past the tracheobronchial regions. Nevertheless, it is estimated that ~20% (6-41%; [42, 43]) of the mass of PM2.5 is in the ultrafine range, and concentrations frequently reach 100 μg/m3 (a high percentage of which will originate from vehicle exhaust) for sustained periods in heavily polluted cities in both developed and developing nations. Furthermore, the airborne mass concentration of 100 μg/m3 represents a moderate/high PM levels over a 24-h period and does not take into account peaks in PM levels that regularly occur in cities, or the increased PM deposition produced by exercise such as brisk walking or cycling in urban environments. In comparison to a long-term continuous inhalation, the instillation regimen will unavoidably induce intermittent very high doses and dose-rate interspersed with long periods of non-exposure . Nevertheless, the similarity of the current results with that of DE inhalation in ApoE−/− mice [10, 11, 19, 20, 45] supports the contention that the biological pathways identified using the instillation protocol are both relevant and important. While a role for the gaseous component of DE cannot be excluded, our results suggest that the particulate component of DE alone is sufficient to enhance plaque formation. It is possible that the gaseous constituents could drive other responses leading to altered plaque composition  which we did not see in our study. The large magnitude of the pro-atherosclerotic effect of DEP will allow us to use this model in the future to explore which attributes and constituents of DEP (e.g. transition metals, polyaromatic hydrocarbons and quinones), and urban PM in general, are responsible for these effects.
Transient exposure to road-traffic has been linked to acute coronary events  and several pre-clinical investigations have suggested that exposure to atmospheric pollution increases markers of lesion vulnerability [8, 10, 11, 27, 29]. Urban PM [8, 27, 30, 47, 48], whole DE [11, 19, 20] or the gaseous components of DE , increase lipid content and inflammatory cells in plaques and the underlying vessel wall. Our results indicated that lipid and inflammatory cells increased in lesions from DEP-instilled mice, but only in proportion to lesion size.
While there was no change in the proportion of individual plaque constituents, or other typical markers associated with plaque vulnerability (e.g. MMPs), the complexity of the lesions in DEP-instilled mice was increased, with more lesions per section and more buried caps within lesions compared with controls. The results are similar to that previously observed in ApoE−/− mice exposed to whole DE by inhalation: an effect that was prevented by addition of a cerium additive to the diesel fuel that decreased the number of particles in the exhaust . It has been suggested that the presence of buried fibrous layers in the brachiocephalic artery are signs of plaque erosion or a previous plaque rupture [49, 50]. The question as to whether buried fibrous layers are evidence of plaque rupture, or is merely a feature of the on-going development of a single plaque, remains a subject of debate [25, 51]. Atherosclerotic lesions in ApoE−/− mice do not rupture catastrophically, but similar changes in human lesions would be associated with increased plaque vulnerability [48, 52]. Thus, these findings would suggest that the ability of DEP to increase plaque size and complexity are likely to be an important contributor to the cardiovascular manifestations of urban air pollution in man.
Previous investigations have suggested that increased atherosclerosis following exposure to DE or CAPs may be due to increased systemic inflammation [11, 27–29], vascular dysfunction [8, 19, 30], or increased oxidative stress [9–11, 30]. In the current investigation, DEP did not increase circulating concentrations of plasma lipids or plaque lipid content, therefore, the exacerbation of lesion formation cannot be attributed to altered lipid handling.
It has been hypothesised that inhaled particles exert their cardiovascular effects indirectly through the passage of inflammatory mediators from the lung to the systemic circulation . DEP caused a clear lung inflammation characterised by infiltration of macrophages and neutrophils into the airways. Interestingly, there was an association with levels of lung inflammation and the size of atherosclerotic plaques. Other studies have failed to find an association between the ability of nanoparticles to induce pulmonary inflammation and their actions on a range of cardiovascular end-points (reviewed in ). Concentrations of acute phase proteins (CRP) in the blood were unchanged by DEP exposure. Although we cannot exclude transient surges in cytokines in response to the particulate, or the involvement of other cytokines (e.g. tumour necrosis factor alpha, interleukins, serum amyloid A3), we have not found consistent changes in any blood cytokine at either early (2 h) or late (24 h) time-points after DE exposure in man [14, 15, 55]. Future studies will address these possibilities in more detail. Additionally, in the present study DEP did not change the inflammatory cell content of atherosclerotic plaques, a finding similar to that after 40 days inhalation of CAPs . Overall, while inflammatory pathways are likely to contribute to the cardiovascular effects of DEP, neither pulmonary nor systemic inflammation alone can account for the atherogenic actions of DEP.
Repeated instillation of DEP was not associated evidence of vascular dysfunction. Previous investigations have reported increased [8, 19] or reduced  vascular contractility accompanying atherosclerosis following pollutant exposure. There have also been contradictory indications of normal  or impaired  acetylcholine-mediated relaxation. However, in the latter, the magnitude of the impairment was small and followed prolonged (>10 weeks) exposure and high-fat feeding. Alternatively, vasomotor impairment may be restricted to resistance arteries  and not the large conductance arteries where atherogenesis takes place. Our results suggest that vascular dysfunction is not required for DEP-induced acceleration of atherosclerosis.
The hepatic up-regulation of several protective antioxidant genes in response to DEP suggests a counter-regulatory response to the systemic pro-oxidative effects of DEP. We have previously shown that the Nfr2 pathway is also upregulated in systemic tissues in response to inhalation of ultrafine urban PM in ApoE−/− mice  and whole diesel exhausts . Interestingly, up-regulation of Nrf2 could trigger vascular proatherogenic effects as we have recently reported that systemic Nrf2 deletion inhibits rather than promotes atherosclerotic lesion formation in the aorta of ApoE null mice . DEP itself can generate free radicals in solution , and oxidative stress is one of the most consistently proposed links between the pulmonary and systemic effects of particulate exposure . Vehicle exhaust promotes lipid peroxidation in plasma lipoproteins and systemic tissues , consistent with recent studies where DE led to increased plasma levels of 8-isoprostanes, 12-HETEs, 13-HODEs, the development of dysfunctional prooxidative and proinflammatory high density lipoprotein (HDL) , and increased isoprostanes in urine . Although we did not evaluate the status of lipid peroxidation in our study, we suggest that the systemic pro-oxidative effects of the particulates in DE could drive a significant portion of the proatherosclerotic actions of urban PM. Interestingly, antioxidant upregulation was especially notable in ApoE−/− mice compared to wild-type mice. This observation adds to the growing evidence that animals/individuals with pre-existing vascular disease (or their risk factors), or diseases with associated cardiovascular complications (e.g. diabetes), may be particularly susceptible to the effects of air pollution [1, 7, 61, 62].