.r. Weeks. 30. 20 lo0. 0. 5. 10. Weeks. Fig. i. Changes in plasma cholesterol and triglyceride. Values are expressed as ..... ROULEAU J.L., PARMLEY W.W., STEVEN J., WILK- ... WILLIS A.J., NAGEL B., CHURCHILL V., WHiiE M.A.,. SMITH D.L. ...
Br. 1. exp. Path. (I988) 69, 515-523
Effects of diltiazem on suppression and regression of experimental atherosclerosis Masahiro Sugano, Yasuhide Nakashima, Hiromi Tasaki, Masayuki Takasugi, Akio Kuroiwa and Osamu Koide* The 2nd Department of Internal Medicine, and the Department of Pathology and Surgical Pathology,* The University of Occupational and Environmental Health Japan, School ofMedicine, i-i Iseigaoka, Yahatanishiku, Kitakyushu, 807, Japan
Received for publication 9 June I 98 7 Accepted for publication 23 February I988
Summary. The effects of diltiazem (a calcium antagonist) on the suppression and regression of atherosclerosis were studied. Thirty-one rabbits were fed a I% cholesterol (atherogenic) diet together with saline (n = 22) or diltiazem (n = 9) injections. After IO weeks, seven rabbits that received saline and nine rabbits that received diltiazem were killed. The remaining I 5 salinetreated rabbits were then put on a standard (regression) diet for the next 1 5 weeks with saline (n = 7) or diltiazem (n = 8) injections. Sixteen rabbits given a standard diet were used as controls. At 5 and IO weeks, the plasma LDL cholesterol level in rabbits on the atherogenic diet with diltiazem was significantly lower than in those on the atherogenic diet with saline. The aortic total cholesterol, esterified cholesterol and calcium contents were also significantly lower in rabbits on the atherogenic diet with diltiazem. After 25 weeks (I5 weeks on the regression diet), the differences in aortic total cholesterol and calcium contents between the two groups on the regression diet were not significant; however, the aortic esterified cholesterol content was significantly lower in the regression diet with diltiazem. The results suggest that diltiazem has a favourable effect both on regression and on suppression of atherosclerosis. Keywords: Ca-antagonist, suppression of atherosclerosis, regression of atherosclerosis, hypercholesterolemic rabbits While several studies suggest calcium antagonists suppress experimental atherosclerosis (Kramsch & Chan 1978, I980; Kramsch et al. I98I; Henry & Bentley I98I; Ginsburg et al. I983; Rouleau et al. I983; Blumlein et al. I984; Willis et al. I985), diltiazem, also a calcium antagonist, has been shown to suppress atherosclerosis in rabbits given a
I% cholesterol diet (Sugano et al. I986). However, the doses used in that study (50 mg daily) were much greater than those used
clinically. In this study, we examined the effects of a pharmacological dose of diltiazem (30 mg) on the suppression and regression of atherosclerosis.
Correspondence: Yasuhide Nakashima, The 2nd Department of Internal Medicine, The University of Occupational and Environmental Health Japan, i-i Iseigaoka, Yahatanishi-ku, Kitakyushu, 807, Japan.
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M. Sugano et al. tein (LDL) and the high-density lipoprotein Materials and methods (HDL) fractions by ultracentrifugation (Brown et al. I969). The HDL supernatant Adult male Japanese white rabbits weighing fraction was obtained by addition ofdextranabout 2.0 kg were used. They were divided Mg2+ to the lower fraction (Warnick et al. into three groups. The i6 control animals I969). Cholesterol values in the plasma, the were fed IOO g of standard pellets (Clea lower fraction, and the HDL fraction were Japan, Inc.) and received one intraperitoneal measured directly; cholesterol values in the injection (i.p.) of saline daily. Twenty-two VLDL and LDL fractions were calculated from animals were fed IOO g of i% cholesterol- those of the plasma, the lower fraction and containing pellets (atherogenic diet) with the HDL fraction. Total cholesterol, free saline i.p. daily. The remaining nine animals cholesterol, and triglycerides were measured were fed the atherogenic diet with diltiazem using enzymatic methods (Wako Pure (30 mg, i.p.) once daily. Chemical Industries, Ltd., Japan) described After weeks, eight of the control rabbits previously (Sugano et al. I986). were killed while the other eight were maintained for a further I5 weeks. Likewise, at Pathological and chemical study of the aorta. weeks, the group of After exsanguination, the aorta between its the end of the first rabbits fed the atherogenic diet with saline origin and the bifurcation of the iliac artery i.p. were divided into three groups, each with was removed, opened longitudinally along comparable levels (mean s.e.) of plasma the midthoracic line and the surface areas cholesterol. containing lesions were measured, without Animals in group (n 7) and all nine staining, as follows; the aorta was covered rabbits on the atherogenic diet with diltia- with clear plastic wrap and the contours and zem were killed at this time (n = 9). Group 2 lesions were outlined in ink. The percentage (n= 7) was put on a standard (regression) of the intima affected by atherosclerosis was diet with saline i.p. Group 3 (n = 8) was fed then determined using a Nikon Cosmozone the regression diet with diltiazem (30 mg) i.p. S-i. Atheromatous lesions were expressed as The regression diet was continued for I5 a percentage of the total aortic surface area. weeks. At the end of the I5 weeks, the After removal of a cross section of the upper rabbits were anaesthetized with 5% pento- thoracic area of each aorta for histological barbital (30-40 mg/kg i.v.), and blood pres- examination, the residual aorta was separsure and heart rate measured using the ated from the adhering adventitial tissue. femoral artery. The rabbits were then killed The intima-media of the aorta was minced by exsanguination from the same artery. into pieces measuring about 2 X 2 mm and Diltiazem was a kind gift from the Tanabe the lipid component extracted twice with Pharmaceutical Company (Tokyo, Japan). chloroform-methanol (2: I, v/v) and stored Food consumption was recorded through- in a dessicator at room temperature until a out the study and the rabbits weighed every constant weight was attained (Nakamura et 5 weeks. al. I 9 71 ). The lipid extracts were used for the measurement of total cholesterol, free choMeasurement of plasma cholesterol and trigly- lesterol, triglycerides and phospholipids. The ceride. Every 5 weeks, after an overnight fast, dried, defatted aortic tissues were assayed for blood samples were drawn from the central calcium, hydroxyproline and uronic acid. The calcium level was measured by atomic ear artery of the rabbits into tubes containing absorption spectroscopy (Model 6o6, PerkinNa-EDTA (I Lg/Ioo pl). The very lowdensity lipoprotein (VLDL) fraction Elmer Corp., Physical Electronics Div., Eden (d. i.oo6) was separated from the bottom Prairie, MN) (Willis I96I; Sugano et al. fraction containing the low-density lipoproI986). About 5 mg of defatted tissue was
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Effect of diltiazem on regression used to assay hydroxyproline according to the method of Stegemann et al. (I958). To measure uronic acid, about 30 mg of dried, defatted tissues were digested with papain (Sigma Chemical Co.), treated with trichloroacetic acid, dialysed and lyophylized according to the method of Nakashima et al. (i98I). Uronic acid was then measured by the carbazole method (Dische I947). The aortic lipid, calcium, hydroxyproline and uronic acid contents are expressed as mg/g of dried, defatted aortic tissue. Statistical analysis was carried out using the Student's t-test. Results Suppression study At 5 and IO weeks the mean plasma LDL cholesterol level of rabbits on the atherogenic diet with diltiazem was significantly
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lower than in those on the atherogenic diet with saline (Fig. i). At 5 weeks, the mean plasma triglyceride level in rabbits on the atherogenic diet with diltiazem was significantly lower than in those on the atherogenic diet with saline, while the plasma HDL cholesterol in the rabbits receiving diltiazem was significantly higher. However, at io weeks, the differences were no longer significant. The VLDL cholesterol levels did not differ between the two groups, either at 5 or IO weeks. Figure 2a shows the outline of representative aortic plaques for each group (the ratios of the lesions to total surface areas were o%, I6% and 13% in the control, saline and diltiazem groups, respectively). The mean ratios of the lesions to total surface area in the diltiazem group were slightly decreased by comparison with the saline group (Fig. 2b). There were no differences in the
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i. Changes in plasma cholesterol and triglyceride. Values are expressed as the mean ± s.e. Assays were carried out at 0, 5 and IO weeks. Significant differences between the two groups on the atherogenic diet are indicated. *P
