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Polycyclic aromatic hydrocarbons induce an inflammatory atherosclerotic plaque phenotype irrespective of their DNA binding properties

Daniëlle M. J. Curfs^*,, Ad M. Knaapen^*, Daniëlle M. F. A. Pachen^*, Marion J. J. Gijbels^,, Esther Lutgens, Marjan L. F. Smook, Mark M. Kockx^||, Mat J. A. P. Daemen and Frederik J. van Schooten^*,1

Departments of
^* Health Risk Analysis and Toxicology,
Molecular Genetics,
Pathology, and Immunology,
University of Maastricht, Maastricht, The Netherlands; and
^|| Department of Pathology, University of Antwerp, Antwerp, Belgium

¹Correspondence: Universiteitssingel 50, Maastricht, Netherlands 6229 ER. E-mail: f.vanschooten{at}grat.unimaas.nl

SPECIFIC AIMS

Polycyclic aromatic hydrocarbons (PAHs) form a large group of widespread environmental pollutants with carcinogenic and atherogenic properties. Whereas the role of PAHs in carcinogenesis has been studied extensively and is believed to involve an initiation-promotion-progression mechanism, the role of PAHs in atherogenesis is less clear. In the current study we aimed to investigate whether DNA binding events in the vessel wall are causally involved in the progression of PAH-mediated atherosclerosis in apolipoprotein E knockout (apoE-KO) mice, thereby answering the question whether the mechanism of PAH-related atherogenesis mimics that of PAH-induced carcinogenesis.

PRINCIPAL FINDINGS

1. B[e]P does not form any detectable levels of DNA adducts
Benzo[e]pyrene (B[e]P) is the non-DNA binding, nonmutagenic structural isoform of the carcinogen benzo[a]pyrene (B[a]P). To confirm that B[e]P does not form any detectable DNA binding products (DNA adducts), DNA adducts were measured in the lung. Results showed that B[e]P indeed did not form any detectable reactive metabolites bound to the DNA (control: <0.1, B[e]P: <0.1, B[a]P: 14.3 ± 0.9 adducts per 10⁸ nucleotides).

2. Both the non-DNA binding, noncarcinogenic PAH benzo[e]pyrene (B[e]P) as well as its DNA binding, carcinogenic structural isomer benzo[a]pyrene (B[a]P) have an accelerating effect on atherosclerotic plaque development
We analyzed the effects of chronic B[a]P and B[e]P exposure (5 mg/kg by oral gavage, once/wk for 24 wk) on number, location, and size of atherosclerotic plaques in the aortic arch of apoE-KO mice. In total, 274 lesions (of which 236 were advanced lesions) were analyzed in the aortic arches of 61 mice: 74 lesions in the B[e]P-exposed animals (n=17), 116 lesions in the B[a]P (n=26), and 84 lesions in the control group (n=18). This means that the number of plaques per arch (4.4, 4.5, and 4.7, respectively) were not affected by the B[e]P or B[a]P exposure compared with the control group. Moreover the location (inner curve of the arch, brachiocephalic trunk, left subclavian artery, and left common carotid artery) of the plaques was not changed upon chronic PAH exposure. Analysis of plaques in the thoracic aorta showed predominantly initial lesions and no difference in the number or size of lesions per thoracic aorta segment between the three groups was found (control: 2.1±0.5, B[e]P: 2.9±0.3, B[a]P: 2.2±0.3, Fig. 1 B).

View larger version (22K): [in this window] [in a new window]   Figure 1. Plaque characteristics of vehicle control, B[e]P, and B[a]P-exposed apoE-KO mice. A) Mean initial and advanced plaque area in the aortic arch, B) mean total plaque area in the thoracic aorta, C) total inflammatory cell (CD45) content, D) T-lymphocyte (CD3) content, E) semiquantitative TGFß1 protein expression. *P < 0.05 vs. control, **2 = 7.0, P < 0.05 vs. control.

We then investigated the size of the atherosclerotic plaques in the aortic arch of apoE-KO mice. Results showed that the size of initial lesions did not change upon PAH exposure, but that the advanced lesions of both B[e]P and B[a]P exposed animals were significantly larger than the controls (Fig. 1A ).

3. Both B[e]P and B[a]P cause an inflammatory atherosclerotic plaque phenotype irrespective of their DNA binding properties
Based on initial results from a previous study, we investigated the effects of both PAHs on inflammatory plaque parameters in the aortic arch. Plaque macrophage content was not significantly different between the B[e]P, B[a]P, and control group (39.4±2.9, 44.6±3.1, and 37.8±4.1%, respectively). However, in both B[e]P and B[a]P-exposed animals, plaque total inflammatory cells (CD45-positive cells) were significantly increased compared with the control group (P<0.05, Fig. 1C ), but no differences were observed between B[a]P and B[e]P. Immunohistochemistry showed a significant increase in plaque T-lymphocytes (CD3 positive cells) in both B[e]P-, and B[a]P-exposed animals compared with the control animals (Fig. 1D ).

4. TGFß expression in macrophages mediates the proinflammatory reaction
As it is an immunomodulating cytokine involved in carcinogenesis as well as atherogenesis, immunohistochemical staining of TGFß1 protein was performed. TGFß1 expression was mainly observed in plaque macrophages. Based on staining intensity, sections of the aortic arches were classified having low or high protein expression. The control animals exhibited predominantly low staining for TGFß1, whereas plaques of the B[a]P and to a lesser extent the B[e]P group were mainly classified as high (B[a]P: ²=7.0, P<0.05. Fig. 1E ).

To investigate the significance of macrophage-specific up-regulation of TGFß in vivo, in vitro experiments were performed using the murine monocyte/macrophage RAW264.7 cell line. Results showed that neutralization of TGFß1 in activated RAW264.7 cells significantly reduced release of the potent atherogenic cytokine TNF (P<0.05). This was paralleled by an inhibiting effect of TGFß antibodies on LPS-induced expression of TNF mRNA. No direct effects of PAHs on release of TNF or expression of TGFß by RAW264.7 cells could be observed.

5. Chronic PAH exposure does not lead to altered levels of blood lipids or circulating inflammatory cells
To exclude PAH-mediated systemic inflammatory effects, we analyzed the blood of the mice on plasma lipid levels and circulating inflammatory cells. Plasma lipid levels were not significantly modulated by the PAH exposure. Moreover, flow cytometry showed no differences in blood T-lymphocytes, B-lymphocytes, granulocytes, and macrophages between B[a]P/B[e]P and control-treated animals. These data indicate that the differences in plaque burden and plaque phenotype could not be explained by differences in inflammatory blood cell composition or plasma lipid levels.

CONCLUSIONS AND SIGNIFICANCE

In the present study, the specific role of PAH-DNA adduct formation in atherosclerosis in apoE-KO mice was investigated by comparing the atherogenic capacity of the adduct-forming carcinogen B[a]P with the chemically almost identical non-adduct-forming, non-carcinogen B[e]P. However, despite the dramatic difference in DNA binding, we showed that both PAHs caused a significant and comparable increase in atherosclerotic plaque progression. Because no DNA adducts could be found in B[e]P-treated animals, these data suggest that DNA adduct formation is rather irrelevant with respect to the atherogenic effects of both B[a]P and B[e]P. This is further substantiated by present and previous data in which we showed that the formation of PAH-DNA adducts was not related to enhanced cell proliferation, expression of p53 protein, or oxidative DNA damage (8-OHdG) in atherosclerotic plaques.

Data from a previous study indicated that chronic B[a]P exposure resulted in a more inflammatory plaque phenotype. To find a possible explanation for the comparable atherogenic effects of both PAHs, we extended these experiments and found that both B[a]P and B[e]P significantly affected the plaque phenotype by causing a profound influx of inflammatory cells into the plaques. For example, exposure to both PAHs significantly increased the T-lymphocyte number in the plaques. In general, the capacity of PAHs to modulate immune cell function has been well documented, and it is suggested that modification of T cell activation could be a specific target in PAH-induced atherogenesis. It has been reported by several groups that depending on the dosage, B[a]P is able to increase or decrease both T- and B-lymphocytes. In our study, using low doses of B[a]P (5 mg/kg wk), an increase of immune cells was found in the plaques in the absence of an increase in circulating macrophages, neutrophils, and B- and T-lymphocytes. This further supports the idea that the observed inflammatory plaque phenotype after PAH exposure is due to a local response of the damaged vessel wall.

To gain more insight in the PAH-induced inflammatory processes in the plaques, we focused on TGFß since it is considered to play a crucial role in the development of atherosclerosis and can be modulated by PAHs. In the present study, we found that TGFß1 protein expression was increased in the plaque macrophages of B[a]P and, though to a lesser extent, also in plaque macrophages of B[e]P-exposed animals. These findings could possibly explain the observed influx of T-lymphocytes into the plaques since it has been reported that TGFß induces the migration of T-lymphocytes. Our in vitro studies using RAW264.7 showed that this induction of TGFß could not be explained by a direct effect of PAHs on the plaque macrophages, suggesting that TGFß expression is merely a consequence of the PAH-mediated ongoing inflammatory changes in the plaques. There is still debate on the role of TGFß1 in vascular disease. Several studies have associated TGFß expression with the development of arterial disease whereas others suggest that TGFß prevents arterial lesion formation. We showed that inhibition of TGFß in activated macrophages was associated with a decreased release of the proatherogenic cytokine TNF. This suggests that the expression of TGFß in plaque macrophages further increases the local proinflammatory effects in the vessel wall and thereby contributes to the enhanced progression of atherosclerosis in the PAH-exposed ApoE-KO mice.

In conclusion, results obtained in this study are in contrast with earlier studies that have tried to link chemical carcinogenic processes (i.e., DNA binding, mutagenesis, proliferation) to atherogenesis. We have shown that the non-adduct-forming B[e]P elicits an inflammatory plaque phenotype in apoE-KO mice comparable to the phenotype induced by the DNA adduct-forming B[a]P. These observations suggest that PAH-DNA adduct formation is not causally involved in PAH-modulated atherogenesis. In contrast, our data indicate that PAHs such as B[a]P and B[e]P might exert their atherogenic effect via stimulation of a general, TGFß-mediated local inflammatory process involving an increased influx of proinflammatory cells into the plaques (Fig. 2 ).

View larger version (17K): [in this window] [in a new window]   Figure 2. Schematic diagram representing a general overview of the proposed mechanism of PAH-mediated atherogenesis in apoE-KO mice. A cross (x) indicates the exclusion of a possible route of action. PAHs are suggested to cause progression of atherosclerosis by inducing a local inflammatory reaction in the vessel wall. Our data further indicate a mediating role of TGFß by enhancing the inflammatory processes in the vessel wall.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2269fje;

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