Ambient particulate pollutants in the ultrafine range promote early atherosclerosis and systemic oxidative stress

Abstract

Air pollution is associated with significant adverse health effects, including increased cardiovascular morbidity and mortality. Exposure to particulate matter with an aerodynamic diameter of <2.5 microm (PM(2.5)) increases ischemic cardiovascular events and promotes atherosclerosis. Moreover, there is increasing evidence that the smallest pollutant particles pose the greatest danger because of their high content of organic chemicals and prooxidative potential. To test this hypothesis, we compared the proatherogenic effects of ambient particles of <0.18 microm (ultrafine particles) with particles of <2.5 microm in genetically susceptible (apolipoprotein E-deficient) mice. These animals were exposed to concentrated ultrafine particles, concentrated particles of <2.5 microm, or filtered air in a mobile animal facility close to a Los Angeles freeway. Ultrafine particle-exposed mice exhibited significantly larger early atherosclerotic lesions than mice exposed to PM(2.5) or filtered air. Exposure to ultrafine particles also resulted in an inhibition of the antiinflammatory capacity of plasma high-density lipoprotein and greater systemic oxidative stress as evidenced by a significant increase in hepatic malondialdehyde levels and upregulation of Nrf2-regulated antioxidant genes. We conclude that ultrafine particles concentrate the proatherogenic effects of ambient PM and may constitute a significant cardiovascular risk factor.

Figure 1

CAP exposures. A, Experimental protocol. Three groups (n=17) of 6-week-old male apoE-null mice were exposed to FA, PM_2.5, and PM of <0.18 μm (UFPs) for 40 days. B and C, Chemical composition of CAPs. UFP air had a greater content of organic and elemental carbon than FP air. Particle chemical composition of the FP (B) and UFP (C) chambers was performed as described in Materials and Methods.

Figure 2

OC composition. Mass concentration fraction of PAHs in the FP (gray) and UFP (black) chambers. Data are shown as nanogram per milligram of PM mass and represent the average of composition analysis performed on filter samples collected for 2 experiments. PAH analysis was performed by means of gas chromatography–mass spectroscopy as described.–

Figure 3

UFP is the most proatherogenic PM fraction. Atherosclerotic lesions were quantitatively analyzed in serial aortic root sections and stained with oil red O. Lesional area was scored as square micrometer per section, averaged ≥25 sections per animal. Group averages are indicated by straight horizontal bars. One FA mouse was an obvious outlier in its group and removed from the atherosclerotic lesion analysis. However, its inclusion did not modify the overall significance. FA mice are represented by dotted circles (n=14), FPs by stripped circles (n=16), and UFPs by filled circles (n=15).

Figure 4

Representative histological photomicrographs. A through C, Oil red O staining for neutral lipids in representative aortic root sections of FA (A), FP (B), and UFP (C) mice. D through F, MOMA-2 immunohistochemical staining in adjacent aortic root sections to those shown in the top row, corresponding to the same FA (D), FP (E), and UFP (F) mice. Both oil red O and MOMA-2 staining yielded red-stained areas. UFP mice exhibited more extensive atherosclerotic plaques (C and F) than FP (B and E) or FA animals (A and D), all consisting primarily of foam cells and macrophages (fatty streaks). Original magnification, ×100.

Figure 5

PM exposure leads to a loss of HDL antiinflammatory properties. Pooled plasma HDL from FA (n=16), FP (n=16) and UFP mice (n=15) was added to cocultures of human artery wall cells in the presence of standard (Std) human LDL, as described in Materials and Methods. Values are expressed as means±SEM of the number of migrated monocytes in 9 fields. Statistical analysis was performed by 1-way ANOVA (Fisher PLSD).

Figure 6

UFP exposure increases liver lipid peroxidation. MDA was assessed in liver homogenates as described in Materials and Methods. Values are expressed as the means±SEM of MDA (nmol/g) in animals from the FA (n=16), FP (n=15), and UFP (n=14) groups. Statistical analysis was performed by 1-way ANOVA (1-tailed Fisher PLSD).

Figure 7

UFP exposure leads to upregulation of antioxidant genes in the liver. mRNA levels for antioxidant genes were determined by quantitative PCR in the livers of chow-fed mice exposed to CAPs for 40 days. Values are expressed as the means±SEM of mRNA levels normalized by β-actin mRNA. Ten samples per group were assayed in duplicate. Statistical analysis was performed by 1-way ANOVA (1-tailed Fisher PLSD; *P<0.05). ATF4 indicates activating transcription factor 4; GST-Ya, glutathione S-transferase Ya; NQO-1, NAD(P)H-quinone oxidoreductase 1; SOD2, superoxide dismutase 2.