Fatty acid oxidation products in human atherosclerotic plaque: an analysis of clinical and histopathological correlates
Introduction
Atherosclerosis is a complex multifactorial disease, believed to be initiated by the uptake of modified low density lipoprotein (LDL) into the arterial wall ultimately resulting in the formation of an atherosclerotic lesion [1], [2]. Over time, development of the lesion can result in plaque fissuring, rupture and thrombus formation, leading in some cases to stroke or myocardial infarction [3], [4].
Much of the recent research on the pathogenesis of atherosclerosis has focused on the involvement of oxidative stress and the possible role of oxidised LDL. In 1979, Goldstein et al. [5] were able to show that oxidised LDL was rapidly ingested by monocytes and appeared to be an initiating factor in the differentiation of monocytes into foam cell macrophages. A role for the participation of such oxidatively modified lipoproteins in atherosclerotic plaque formation is suggested by studies showing the presence of antibodies specific to oxidised LDL in atherosclerotic plaque [6], as well as products resulting directly from oxidation of LDL such as F2-isoprostanes and hydroxyeicosanoids [7], [8], [9], [10].
The F2-isoprostanes, free radical oxidation products of arachidonic acid, are a specific and widely accepted method for measurement of in vivo lipid peroxidative stress [11], [12]. However, it has been shown that primary oxidative products of linoleic and arachidonic acid are more abundant in atherosclerotic plaque than F2-isoprostanes [13]. Mallat et al. [9], found that hydroxyeicosatetraenoic acids (HETEs), another family of compounds resulting from oxidation of arachidonic acid, were present in levels almost 24 times higher than F2-isoprostanes in atherosclerotic plaque, and that the level of HETEs was more than 70% higher in unstable versus stable plaques, i.e. those obtained from patients with recent symptoms of cerebrovascular disease. We have recently confirmed that both HETEs and compounds resulting from oxidation of linoleic acid (LA), such as hydroxyoctadecaenoic acids (HODEs) and oxo-octadecaenoic acids (oxoODEs), are present in quantifiable amounts in atherosclerotic plaque, and have developed a convenient HPLC method for their simultaneous quantitation [10].
While these compounds may represent markers of in vivo oxidative damage, they also possess biological activities which may be relevant to atherogenesis. The adherence of red blood cells to the endothelium, activation of inflammatory mediators and differentiation of monocytes are all reported biological activities of oxidised fatty acid products [14], [15], [16]. Symptoms of atherosclerosis, are often associated with the formation of thrombi, usually the result of rupture of an unstable plaque [17]. According to Stary et al. [18], lesions that are most at risk of disruption and rupture are those that are predominantly lipid containing. This may in part, be due to the presence of inflammatory cells, toxic products resulting from proteolytic degradation by enzymes or an overabundance of macrophage foam cells [19], [20]. With many of the fatty acid oxidation products possessing biological activities that promote such processes, it is possible that they may be important cellular mediators in plaque development and instability.
Hydroxy fatty acids can be formed by non-enzymatic oxidation of polyunsaturated fatty acids as well as by enzymes such as lipoxygenases. The formation of 5-, 12- and 15-HETE have been shown to be the result of 5-, 12- and 15-lipoxygenase, respectively, [21]. The lipoxygenases have been found to be expressed in human atherosclerotic arteries [22] and appear to be active in early stages of plaque development [23], [24]. However, chiral analysis suggests that hydroxy fatty acids found in advanced plaque are formed predominantly by non-enzymatic processes [10], [23]. Therefore, identification and quantitation of certain fatty acid oxidation products may provide an insight into the processes at work at different developmental stages of the disease.
Atherosclerosis, a disease of complex etiology, has been reported to be associated with many risk factors including cholesterol and lipid levels, blood pressure, diabetes, obesity, smoking and excessive alcohol intake [25], [26], [27], [28]. Some of these risk factors may be connected through the common mechanism of increasing oxidative stress. The aim of the present study was to quantitate the major fatty acid oxidation products in human carotid artery atherosclerotic plaque to determine if there was a relationship between levels of these compounds and the presence or absence of recent cerebrovascular symptoms to test the hypothesis that specific HETEs or HODEs may be markers of plaque instability [9]. We also examined if there was any correlation between the observed levels of oxidation products with plaque histopathology. Using clinical characteristics, the associations between atherosclerotic risk factors and levels of fatty acid oxidation products and F2-isoprostanes were also investigated.
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Chemicals and reagents
Prostaglandin B2 and 8-iso-prostaglandin F2α-d4 were purchased from Caymen Chemical Company (Ann Arbor, MI). All solvents used in the chromatography were of HPLC grade. The derivatising reagents N,O-bis (trimethylsilyl) trifluoroacetamide+1% trimethylchlorosilane (BSTFA+1% TMCS) was purchased from Pierce Chemicals (Rockford, IL), pentafluorobenzylbromide (PFBBr) and N,N-diisopropylethylamine (DIPEA) were purchased from Sigma Chemicals (St Louis, MO) while anhydrous pyridine was obtained from
Patient characteristics
A total of 50 atherosclerotic plaque samples was obtained for analysis. Three patients were excluded on the basis of evidence of pre-existing chronic renal failure and their data were not used in the analysis. We were unable to review the patient records for a further case and, therefore, utilised that data only in the analysis of plaque histopathology. Using the predefined criteria, 29 patients were identified as symptomatic and 17 as asymptomatic for cerebrovascular disease. Table 1 outlines
Discussion
The aim of this investigation was to examine plaque tissue levels of fatty acid oxidation products in relation to cerebrovascular symptoms, carotid plaque histopathology, and atherosclerotic risk factors. We were able to quantify a range of fatty acid oxidation products and demonstrate that they are formed predominantly by non-enzymatic processes. There were no differences in the levels of any of the identified oxidation products between plaque histopathological sub-types. While there was a
Acknowledgements
We gratefully acknowledge the support of the theatre nursing staff at Royal Perth Hospital. This study was supported by grant no. 9937262 from the National Health and Medical Research Council of Australia (9937262) and through post-graduate research funding from the University of Western Australia (Emma Waddington).
References (45)
- et al.
The role of oxidised lipoproteins in atherosclerosis
Free Radicals in Biology and Medicine
(1996) - et al.
Identification and quantitation of unique fatty acid oxidation products in human atherosclerotic plaque using high-performance liquid chromatography
Analytical Biochemistry
(2001) - et al.
Indices of lipid peroxidation in vivo: strengths and limitations
Free Radical Biology and Medicine
(2000) - et al.
Measurement of F2-isoprostanes as an index of oxidative stress in vivo
Free Radical Biology and Medicine
(2000) - et al.
Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development
Biochimica, Biophysica Acta
(1995) - et al.
Stimulation of human neutrophils by 5-oxo-6,8,11,14-eicosatetraenoic acid by metabolism independent of the leukotriene B4 receptor
Journal of Biological Chemistry
(1993) - et al.
Hydroxyeicosatetraenoic acids (HETEs)
Progress in Lipid Research
(1988) - et al.
Oxidation of LDL by rabbit and human 15-lipoxygenase: prevalence of nonenzymatic reactions
Journal of Lipid Research
(2001) Update on the role of cigarette smoking in coronary artery disease
American Heart Journal
(1981)- et al.
An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry
Analytical Biochemistry
(1999)