Elsevier

Atherosclerosis

Volume 175, Issue 2, August 2004, Pages 203-212
Atherosclerosis

Endothelin-1(1–31) levels are increased in atherosclerotic lesions of the thoracic aorta of hypercholesterolemic hamsters

https://doi.org/10.1016/j.atherosclerosis.2003.10.015Get rights and content

Abstract

Objective: The novel vaso-constricting 31-amino acid-length endothelin-1 [ET-1(1–31)] is selectively produced by human mast cell chymase via its action on big ET-1. However, the pathological role of ET-1(1–31) in atherosclerosis remains unclear. The aim of this study was to clarify vasoconstrictive response and expression of ET-1(1–31) in atherosclerotic aorta. Methods and results: Syrian golden hamster, was used for preparing the atherosclerotic models by the administration of a high cholesterol diet (HC), treatment with the nitric oxide synthase inhibitor (Nω-nitro-l-arginine methylester, l-NAME) alone, or both (HC and l-NAME) for 40 weeks. Early atherosclerosis was observed in the case of HC or l-NAME alone treatments respectively and severe atherosclerosis was observed in the case of combined HC and l-NAME treatment. Vasoconstriction induced by ET-1(1–31) was not altered by the atherosclerotic changes, but the expression pattern of ET-1(1–31) was different at each stage of the atherosclerotic aorta. ET-1(1–31) was observed rarely in normal aortas or in early atherosclerotic lesions, but ET-1(1–31) expression was dramatically increased in aortic neointima and adventitia in a state of atherosclerosis with severe inflammation. Conclusion: ET-1(1–31) might play in a role of promoting atherosclerosis, and especially be involved in inflammatory mediation during the progression of atherosclerosis.

Introduction

Endothelin (ET)-1(1–21), a 21-amino acid peptide produced from big ET-1 hydrolysis by the ET converting enzyme [1], was isolated from the supernatant of cultured porcine coronary artery endothelial cells as a bioactive peptide that has constrictive activity with respect to smooth muscle [2]. ET-1(1–21) plays an important role in the maintenance of basal vascular tone and has been implicated in the pathophysiology of vasospastic reactions [3]. In addition, ET-1(1–21) has mitogenic properties on vascular smooth muscle cells (VSMCs) and ET-1(1–21)-like immunoreactivity had been observed in atherosclerotic coronary plaques [4]. Moreover, increased ET-1 mRNA expressions had been reported in atherosclerotic carotid lesions [5]. Thus, the possibility that ET-1(1–21) might play an important role in the development of atherosclerosis cannot be excluded.

We previously reported that 31-amino acid endothelins [ETs(1–31)] are formed via cleavage of big ETs at the Tyr31–Gly32 bond by human mast cell chymase or other chymotrypsin-type proteases [6]. ET-1(1–31) exerts a vasoconstricting action, the activity of which is about 10 times weaker than that of ET-1(1–21) in the porcine coronary artery [7]. Our recent investigations of porcine coronary artery demonstrated that ET-1(1–31) is involved in VSMC proliferation similar to that of ET-1(1–21) [8]. These findings suggest that the ET-1(1–31) may be associated with atherosclerosis. However, the pathophysiological role of ET-1(1–31) in atherosclerosis remains unclear.

A hamster model was used to investigate the role of chymase-induced ET-1(1–31) in atherosclerosis, since the specificity and activity of hamster chymase is similar with that of human [9]. This hypercholesterolemic hamster model, where atherosclerosis is induced by a high cholesterol/fat diet, is useful for research on an early pro-atherogenic event, namely, subendothelial monocyte-macrophage foam cell formation [10]. However, the drawback of this hamster model is that the histological changes associated with atherosclerosis usually remain in this early stage for periods of up to 12 months [11]. Hypercholesterolemia, smoking, hypertension, and diabetes mellitus are important risk factors for atherosclerosis, which are all associated with reduced nitric oxide (NO)-dependent vasodilation, even before the development of clinically or morphologically apparent atherosclerosis [12]. The importance of NO in the development of atherosclerosis is supported by the observation that endogenously formed NO may also protect against the formation of foam cells and media hypertrophy, i.e. against the structural component of atherosclerosis [13]. Thus, to enhance the severity of the atherosclerotic lesions in the hypercholesterolemic hamster, we have here investigated the effect of long-term inhibition of NO production in the hypercholesterolemic hamster model on atherosclerotic progression.

In the present study, we functionally and pathophysiologically correlated ET-1(1–31) with atherosclerosis by measuring vasoconstriction in atherosclerotic arteries and immunohistochemical changes in ET-1(1–31) with the progression of atherosclerosis, using early or advanced stages of atherosclerosis in the hamster model.

Section snippets

Animals, diet and experimental groups

Twenty-four Syrian golden, specific pyrogen-free male hamsters, aged 5 weeks, were obtained from Japan SLC (Shizuoka, Japan). After 1 week of adaptation, the hamsters were randomly divided into four subgroups as follows. SD: standard diet with normal tap water, SD/NAME: standard diet with 1 mg/ml Nω-nitro-l-arginine methylester (l-NAME) in the drinking water, HC: diet supplemented with 0.3% cholesterol (w/w) and 10% coconut oil (w/w) (Oriental Yeast, Osaka, Japan) as high cholesterol diet with

Biochemical and physiological measurements

Table 1 shows the body weight, heart weight, serum lipid values, systolic blood pressure, and heart rate in each experimental. There were no differences in the above parameters among the groups at the beginning of the experiments. Increases in body weight were not significantly different between each group. The intake of cholesterol during the experiments was the same for the HC and HC/NAME groups, and the intake of l-NAME was the same for the SD/NAME and HC/NAME groups. Serum levels of total

Atherosclerosis models

We sought to develop a hamster model of severe atherosclerosis by feeding hamsters with a high cholesterol diet in combination with l-NAME, an inhibitor of NO production (HC/NAME group). As controls, we also fed other hamsters with high cholesterol diets alone (HC group) or l-NAME alone (SD/NAME group). These latter models showed signs of early atherosclerosis [14], [15], including the thickening of the intima and/or macrophage-foam cell formation in their aortic arches. Furthermore, the HC

Acknowledgements

The excellent technical assistance of Mr. Yoshimichi Endo is gratefully acknowledged. This study was supported in part by Sasagawa grants (no. 12-160K).

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