Acrolein activates matrix metalloproteinases by increasing reactive oxygen species in macrophages

Abstract

Acrolein is a ubiquitous component of environmental pollutants such as automobile exhaust, cigarette, wood, and coal smoke. It is also a natural constituent of several foods and is generated endogenously during inflammation or oxidation of unsaturated lipids. Because increased inflammation and episodic exposure to acrolein-rich pollutants such as traffic emissions or cigarette smoke have been linked to acute myocardial infarction, we examined the effects of acrolein on matrix metalloproteinases (MMPs), which destabilize atherosclerotic plaques. Our studies show that exposure to acrolein resulted in the secretion of MMP-9 from differentiated THP-1 macrophages. Acrolein-treatment of macrophages also led to an increase in reactive oxygen species (ROS), free intracellular calcium ([Ca²⁺]_i), and xanthine oxidase (XO) activity. ROS production was prevented by allopurinol, but not by rotenone or apocynin and by buffering changes in [Ca²⁺]_I with BAPTA-AM. The increase in MMP production was abolished by pre-treatment with the antioxidants Tiron and N-acetyl cysteine (NAC) or with the xanthine oxidase inhibitors allopurinol or oxypurinol. Finally, MMP activity was significantly stimulated in aortic sections from apoE-null mice containing advanced atherosclerotic lesions after exposure to acrolein ex vivo. These observations suggest that acrolein exposure results in MMP secretion from macrophages via a mechanism that involves an increase in [Ca²⁺]_I, leading to xanthine oxidase activation and an increase in ROS production. ROS-dependent activation of MMPs by acrolein could destabilize atherosclerotic lesions during brief episodes of inflammation or pollutant exposure.

Introduction

Atherosclerotic disease leading to myocardial infarction (MI) and stroke is the leading cause of morbidity and mortality in the Western world (Rosamond et al., 2008). It is currently believed that atherosclerotic lesions develop and grow as a result of vascular inflammation that leads to monocyte infiltration into the vessel wall. Once lodged in the sub-intimal space, the monocytes differentiate into macrophages which take up modified lipoproteins and as a result are transformed into foam cells (Glass and Witztum, 2001, Reiss and Glass, 2006). These processes lead to the formation of distinct lesions (plaque) which are composed of foam cells, a lipid-rich-core and a fibrous, matrix-rich cap. Gradual erosion of this cap or its acute rupture exposes platelets to the underlying matrix proteins and pro-thrombotic molecules, activating them and initiating a thrombotic response. Vascular occlusion at these sites initiates ischemic episodes associated with myocardial and cerebral infarction.

Although physiological events contributing to plaque erosion and rupture are complex, matrix metalloproteinases (MMPs) have been suggested to be play an important role (Galis et al., 1994, Galis et al., 1995, Herman et al., 2001, Sukhova et al., 1999). MMP-mediated degradation of the extracellular matrix (ECM) is vital for several physiological functions including development, morphogenesis, angiogenesis, and tissue repair. Several pathological conditions, such as arthritis, cancer, nephritis, chronic ulcers, and fibrosis (Nagase et al., 2006) are, however, associated with excessive or unregulated MMP activity. MMP dysregulation is also a characteristic feature of cardiovascular abnormalities and increased levels of MMPs have indeed been found in atherosclerotic plaques (Galis et al., 1994, Galis et al., 1995, Halpert et al., 1996, Rajavashisth et al., 1999) and in patients with unstable angina or acute myocardial infarction (Kai et al., 1998, Tziakas et al., 2004).

Acrolein is an aldehydic compound that has been linked in epidemiological studies to cardiovascular pathology (Bhatnagar, 2004, Feron et al., 1991). It is a particularly abundant component of air-borne particulate matter (PM) that arises during the burning of fossil fuels, cigarettes, or other organic material. In addition, acrolein is generated during the cooking or frying of food, is present in the effluent of industrial waste, and exists naturally in vegetables, fruits, and herbs (Feron et al., 1991). Acrolein is also an end product of the metabolism of certain pharmaceuticals (Ludeman, 1999) and can likewise be produced by myeloperoxidase-catalyzed oxidation. It is therefore generated in high amounts at sites of inflammation (Anderson et al., 1997) or lipoprotein oxidation (Anderson et al., 1997, Burke et al., 2001, Feron et al., 1991, Steinberg, 1997), particularly within vascular lesions (Shao et al., 2005). The toxicity of acrolein is a consequence of its strongly reactive, electrophilic, carbonyl group which can react with cellular nucleophiles such as thiols or amines (Esterbauer et al., 1991). Thus acrolein can form adducts with proteins, disrupting cellular signaling or function, or nucleic acids, eliciting mutagenic or carcinogenic effects. In addition, the toxic effects of acrolein can result from indirect means. In particular, chronic acrolein exposure could deplete cellular antioxidants such as glutathione, rendering the cell prone to damage from free radicals. Cardiovascular tissue seems to be particularly sensitive to the toxic effects of acrolein. Epidemiological and animal studies have linked acrolein exposure to arrhythmia (Bhatnagar 1995), hypertension (Feron et al., 1991), atherogenesis (Steinbrecher et al., 1990), dyslipidemia (McCall et al., 1995), and myocardial infarction (Alfredsson et al., 1993, Levine et al., 1984, Stewart et al., 1990). Despite these associations however, the precise mechanisms whereby acrolein contributes to acute cardiovascular pathology is unknown.

Given the extensive evidence implicating oxidative stress in MMP activation (Nelson and Melendez, 2004), we tested the hypothesis that acrolein exposure results in MMP activation and thus contributes to acute plaque rupture and vascular occlusion. Using both a cell culture model and murine atherosclerotic tissue, we did indeed demonstrate that one consequence of acrolein exposure is MMP secretion. Furthermore we show that this was dependent upon increased intracellular calcium and increased ROS generation by xanthine oxidase. Our findings are likely to be of significance in understanding the acute inflammatory responses to acrolein generated endogenously or delivered from the environment.