Inflammatory Response in the Pathogenesis of Atherosclerosis and Its

Chapter 18

Inflammatory Response in the Pathogenesis of Atherosclerosis and Its Prevention by Rosmarinic Acid, a Functional Ingredient of Rosemary 1

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Y u j i Naito , Shigenori Oka , and Toshikazu Yoshikawa

Downloaded by UNIV OF ARIZONA on June 11, 2017 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch018

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First Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Research and Development Center, Nagase Company, Ltd., Kobe 651-2241, Japan 2

Recent evidence suggests a role for the inflammatory response in the pathogenesis of atherosclerosis with the adhesion of circulating monocytes to the endothelium at site of injury. Leukocytes adhere and immigrate into the subendothelial space in response to chemoattractants and other activating molecules, which are mediated by adhesion molecules located on leukocyte/endothelial cells. Oxidatively modified low -density lipoprotein is thought to play a key role in these reactions. We recently found that the expression of adhesion molecules was upregulated by oxidized lipoprotein or inflammatory cytokines, and that the increased expression was inhibited by α-tocopherol as well as by functional ingredients of food factors such as rosmarinic acid. Rosmarinic acid has potent free radical scavenging and anti-inflammatory activities, and its administration reduced the serum levels of lipid peroxides and inhibited the progression of atherosclerosis in apolipoprotein Ε-deficient mice without affecting the serum level in cholesterol. These anti-inflammatory properties of this food factors suggest that they may have potential benefits i n atherosclerosis. There is considerable evidence that inflammatory response induced by oxidized low-density lipoprotein (oxLDL) contribute to the development of atherosclerosis. O x L D L or their metabolites have been found in atherosclerotic lesions in both human and animal models (1). OxLDLs have been shown to stimulate endothelial cells to express several proteins that contribute to atherosclerosis, including monocyte chemotactic protein-1 (MCP-1), macrophage-colony-stimulating factor (M-CSF), vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule 1 (ICAM-1) (2,3). O x L D L is avidly taken up by macrophages, resulting in foam cell formation (4). 208

© 2003 American Chemical Society Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


209 The significant role of adhesion molecules or macrophages in the process of atherosclerosis was recently confirmed by molecular biological techniques, in which knockout of ICAM-1 gene (5) or administration with monoclonal antibody against the receptor M-CSF (6) significantly inhibited the progression of atherosclerosis in apolipoprotein Ε-deficient mice. The expression of adhesion molecules ICAM-1 and V C A M - 1 was upregulated by o x L D L or inflammatory cytokines, and that the increased expression was inhibited by α-tocopherol (7). Rosemary is one of the world's best-known spices, and it has also been universally used throughout the history for its health benefits. The dried extracts of rosemary contain caffeic acid, chlorogenic acid, rosmarinic acid, carnosol, carnosic acid, and ursolic acid. Rosmarinic acid is a major phenolic ingredient of rosemary (8). In this study, we investigated the antioxidative and anti inflammatory activities of rosmarinic acid in vitro, and effects of its administration on inhibiting of progression of atherosclerosis in apolipoprotein Ε-deficient mice. Free Radical scavenging activity Superoxide and hydroxyl radical scavenging activities were measured by the electron paramagnetic resonance (EPR) spin trapping method. Following previous reports (9,10), superoxide and hydroxyl radicals generated by the hypoxanlhine-xanthine oxidase enzyme system and the Fenton system, containing F e S 0 and hydrogen peroxide, respectively, were trapped by 5,5dimethyl-1 -pyrroline-N-oxide (DMPO). Rosmarinic acid inhibited the signal intensity of D M P O - O O H (spin adduct trapping superoxide) as well as that of D M P O - O H (spin adduct trapping hydroxyl radical) in a concentration-dependent manner with I C concentrations of 0.62 μΜ for D M P O - O O H and 15 μΜ for DMPO-OH. 4

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Inhibition of L D L oxidation Human L D L was incubated at 37 °C with 5 m M each azo-compound, 2, 2'azobis-(2-amidinopropane)hydrochloride (AAPH) or 2,2'-azobis(2,4dimethylvaleronitrile) ( A M V N ) , in the absence or presence of different concentrations of rosmarinic acid. Lipid peroxides were determined by ferrous oxidation in xylenol orange assay (FOX-assay). Rosmarinic acid significantly inhibited the increase in lipid peroxides induced by A A P H in concentrations of 1 and 10 μΜ, and slightly inhibited those induced by A M V N at a dose of 10 μΜ. Effects of rosmarinic acid on adhesion molecule expression induced by oxLDL The L D L (d=1.019 - 1.063) was isolated from the serum of healthy human donors by density-gradient ultracentrifugation, dialyzed with phosphate buffered saline containing E D T A , and stored at 4°C. Lipoprotein concentrations were expressed as protein content. O x L D L was prepared by incubating L D L with 10 μΜ F e S 0 at 37°C for 30 min. The oxidation of L D L produced 50 - 100 n M 4

Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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hydroperoxide per mg protein of L D L . Enzyme immunoassay (EIA) was used to assess the adhesion molecule expression on the human aortic endothelial cells (HAEC) monolayer. Confluent H A E C monolayers were pretreated with and without rosmarinic acid for 1 h, and then H A E C were stimulated with oxLDL (100 μg/ml). Rosmarinic acid significantly inhibited the increase in the ICAM-1 expression in a concentration-dependent manner (Fig.l). V C A M - 1 expression was also increased by stimulation of oxLDL and the increase was significantly inhibited by rosmarinic acid in a concentration-dependent manner.


Effect of rosmarinic acid on monocyte adhesion to H A E C The H A E C monolayers prepared in 48-well plates were stimulated with interleukin-ΐβ (IL-Ιβ, 20 u/ml) for 4 h in the presence or absence of rosmarinic acid. Subsequently, H A E C were washed with Hanks buffered salf solution, and monocytes (U937 cells) were added to each well and incubated at 37°C for 30 min. U937 cell adhesion was determined by visual counting under a phasecontrast microscope. The adherence of monocytes to H A E C exposed to ILΙβ was siginificantly increased compared to that of unstimulated H A E C . Figure 2 shows the effect of rosmarinic acid on the adherence of monocyte to IL-Ιβstimulated H A E C . The treatment of H A E C with rosmarinic acid significantly reduced the adherence of monocyte on H A E C in a concentration-dependent manner. Rosmarinic acid attenuates development of atherosclerosis i n apolipoprotein Ε-deficient mice The ex vivo effect of rosmarinic acid on the development of atherosclerosis in apolipoprotein Ε-deficient mice, in relation to plasma cholesterol and lipid peroxide levels, was investigated. Apolipoprotein Ε-deficient mice (4-week-old) were divided into two groups fed for 6 weeks via their drinking water with the following: control group and rosmarinic acid group (0.04 % solution, 1.52 mg/kg/day). The aortic sections were made and stained with Oil-red-O, and counter-stained with hematoxylin. Average lesion sizes of 7-9 sections were measured by quantitating the lesion-covered areas on the aortic vessel walls (NIH image 1.62 program; National Institute of Health). Aortic atherosclerotic lesion areas were reduced in mice that consumed 1.52 mg of rosmarinic acid /kg/day compared to mice on the control diet. Figure 3 shows the photomicrographs of typical atherosclerotic lesion of the aortic arch of apolipoprotein Ε-deficient mice after treatment. Rosmarinic acid inhibited plasma lipid peroxide levels in apolipoprotein Ε-deficient mice without affecting the total cholesterol levels in plasma, indicating that the anti-atherosclerotic properties of rosmarinic acid may be due, in part, to its antioxidative action which inhibits the increase in lipid peroxides.

Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Figure 1. Effect of rosmarinic acid on ICAM-1 expression stimulated with oxLDL. Confluent HAEC were incubated with ox-LDL (100 yg/mL) in the absence or presence of rosmarinic acid at 37 C for 6 h. The expression of ICAM-1 on the cell surface was measured using EIA. *p

Inflammatory Response in the Pathogenesis of Atherosclerosis and Its

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