Anti-atherogenic effect of BHB-TZD having inhibitory activities on cyclooxygenase and 5-lipoxygenase in hyperlipidemic mice
Introduction
Atherosclerosis is a complex and chronic inflammatory disease involving elastic and muscular arteries [1]. The chronic inflammatory response of the vascular wall is thought to involve uncontrolled proliferation of vascular myointimal cells, production of foam cells, and progressive vascular occlusion [2]. The inflammatory cells in atherosclerotic lesions produce several kinds of matrix degrading enzymes, which are important in plaque stability.
Arachidonic acid metabolites, including prostaglandins (PGs), thromboxanes (Txs), and leukotrienes (LTs), are generated from arachidonic acid primarily by two enzymes, cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). These metabolites have been shown to play important roles in inflammation, thrombosis, allergy and cancer. There are two types of COX, the constitutive enzyme COX-1 and the inducible enzyme COX-2, both of which are present in atherosclerotic lesions [3]. These enzymes produce various prostanoids, including PGE2 and TxA2, which have been shown to have proatherogenic effects [4], [5]. 5-LOX has a key role in the production of leukotrienes, such as LTA4, LTB4 and LTD4, which have proinflammatory activity and are involved in the pathogenesis of various inflammatory disorders [6]. The number of 5-LOX expressing cells, primarily macrophages, is markedly increased in advanced atherosclerotic lesions [7], and a polymorphism in the 5-LOX promoter has been linked to atherosclerosis [8]. Various inhibitors of COX-1, COX-2, or both, and suppression of the 5-LOX pathway by genetic ablation or receptor antagonists, have been reported to reduce the size of atherosclerotic lesions by reducing the production of inflammatory prostanoids, such as PGE2 and TxA2, or leukotrienes, such as LTB4 [5], [9], [10]. The involvement of COX and the 5-LOX pathway in the stability of atheromatous plaques, however, has not yet been determined.
Monocyte prostaglandins have been shown to inhibit procollagen secretion by human vascular smooth muscle cells [11]. Moreover, PGE2 produced by functionally coupled COX-2 and PGE2 synthase in human atherosclerotic lesions was found to increase the production of matrix metalloproteinases (MMPs), thus contributing to plaque instability and suggesting that COX inhibitors are involved in plaque stabilization [12]. In addition, 5-LOX deficient mice have been shown to have a marked reduction in the formation of aortic aneurysms, which is associated with reduced MMP-2 activity [13]. In humans, 5-LOX expression is higher in symptomatic than in asymptomatic plaques, an activity associated with acute ischemic syndrome, possibly through the generation of LTB4 and subsequent MMP biosynthesis [14]. These findings suggest that both COX and 5-LOX may play pivotal roles in plaque instability and that the use of dual inhibitors of both enzymes may be an effective strategy for increasing plaque stability. These dual inhibitors may also be a promising new therapeutic regimen for atherosclerosis, since they have fewer adverse effects than existing selective or combined COX inhibitors [15]. Vidal et al. have been demonstrated that licofelone, a dual COX and 5-LOX inhibitor, attenuated atherosclerosis in rabbit atherosclerosis model [16].
One of these dual COX and 5-LOX inhibitors, BHB-TZD [5-(3,5-di-tert-butyl-4-hydroxybenzylidene)thiazolidin-2,4-dione] has been found to reduce the number of lesions in adjuvant induced polyarthritis, a chronic model of inflammation, with least gastric ulceration [17], [18]. We have therefore investigated the effect of BHB-TZD on atherosclerosis in LDLR−/− mice, including its effects on lesion formation and plaque phenotype.
Section snippets
Animal model and treatment with BHB-TZD
BHB-TZD was obtained from the Korea Research Institute of Bioscience and Biotechnology (see Supplementary Fig. I). Eight-week-old male LDL receptor knock-out (LDLR−/−) mice on a C57BL/6 background (n = 30) were randomly divided into two equal groups. The BHB-TZD group was fed a western diet (CRF-1 supplemented with 0.15% cholesterol, 20% fat, and 0.05% Na-cholate, Oriental Yeast Co. Ltd., Japan) plus 0.1% (w/w BHB-TZD), and the control group was fed a western diet alone. The western diet
The plasma levels of BHB-TZD and arachidonic acid metabolites
The pharmacokinetics of BHB-TZD in LDLR−/− mice was determined after oral administration of 3 mg of BHB-TZD, equivalent to the dietary intake of BHB-TZD each day (Supplementary Fig. II), and blood samples were collected at 12, 24, and 48 h. The plasma level was increased up to 13.9 ± 3.7 μg/ml at 12 h, and decreased to 7.1 ± 5.5 μg/ml at 24 h and could not be detected at 48 h. According to previous studies [17], [18], these plasma levels of BHB-TZD are beyond the IC50 for COX-1, 2 and 5-LOX. To determine
Discussion
We have shown here that BHB-TZD significantly ameliorates the formation of typical atheromatous lesions in LDLR−/− mice, and found that BHB-TZD reduced the overall plasma concentrations of prostanoids and LTB4 and triglycerides. BHB-TZD also reduced the number of macrophages infiltrating into early atheromatous lesions of the aortic arch. These findings were supported by results showing that aortic VCAM-1 and MCP-1 expression was lower in BHB-TZD than in control mice. We also found that BHB-TZD
Acknowledgment
This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A090264).
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Current address: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, USA.