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Effect of metoprolol on plasma lipids and arterial intimal lipid deposition in spontaneously hypertensive rats
Atherosclerosis, 80 (1989) 135-142 Elsevier Scientific Publishers Ireland.
ATHERO
135 Ltd.
04408
Effect of metoprolol on plasma lipids and arterial intimal lipid deposition in spontaneously hypertensive rats A.-M. hstlund-Lindqvist
I, A. Eklund
*, L. Sjablom
* and L. Jijnsson
3
’Departmeni of Biochemistry, Hksle Research Laboratories, Miilndal, ’ Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala and ’ Department of Pathology, Swedish University of Agricultural Sciences, Uppsala (Sweden) (Received 25 January, 1989) (Revised, received 21 August, 1989) (Accepted 24 August, 1989)
Summary The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in spontaneously hypertensive rats (SHR) and to reveal any influence of treatment with metoprolol on these parameters. Metoprolol treatment caused an 8% decrease in heart rate and a 13% decrease in blood pressure and led to a rise in plasma triglycerides, 24%, 17% and 34% after 1, 3 and 6 months of metoprolol treatment, respectively. However, no effect on plasma triglycerides was observed after 9 months of metoprolol treatment while a reduced cholesterolemic response was observed. Intimal proliferations containing accumulations of lipids were observed in small intramural branches of coronary arteries (> 100 pm) in 11 of 31 control rats fed the atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-treated rats fed an otherwise identical diet. The corresponding figures for the frequency of lipid containing intimal plaques in aorta were 6/19 in controls and 2/24 in the metoprolol-treated group.
Key words:
Atherosclerosis; Metoprolol
Aorta;
Coronary
arteries;
Introduction In previous studies, non-selective P-blockers have been shown to have an anti-atherogenic ef-
Correspondence to: A.-M. ijstlund-Lindqvist, Ph.D., Department of Biochemistry, HBssle Research Laboratories, S-431 83 Miilndal, Sweden. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
Lipids;
Spontaneously
hypertensive
rats;
feet in cholesterol-fed rabbits [l-3]. Recently, the same effect has been demonstrated for the adrenergic &-receptor antagonist, metoprolol [4,5]. The mechanism underlying this effect is not known. In order to test the species-specificity of the phenomenon, we wanted to extend the studies of the anti-atherogenic effect of metoprolol by looking for possible influences of metoprolol in a different animal species. Ltd.
136 In contrast to the rabbit, which has been the most frequently used experimental animal in atherosclerosis research, the rat has been considered very resistant to atherosclerosis. However, rats develop atherosclerosis as they grow old [6] and certain strains of rat, such as the LA/N corpulent rat [7], develop atherosclerosis within about 9 months. Sclerotic lesions, consisting of raised intimal plaques in the aorta, have also been found in all male Sprague-Dawley-strain rats reaching a plasma cholesterol level above 3.9 mmol/l in a study extending over 80 weeks [8]. Furthermore, the stroke-prone spontaneously hypertensive rats (SHRSP) [9] rapidly develop arterial fat deposits when fed on high cholesterol diets. As both hypertension and hyperlipidemia are well-known risk factors for the development of atherosclerosis, we decided to test the effect of metoprolol treatment on atherosclerosis and on plasma levels of lipids in spontaneously hypertensive male rats (SHR) fed an atherogenic diet. Materials and methods Experimental design Seventy male, inbred, spontaneously hypertensive SHR/ Molrats (Mollegaard, Ltd., Skensved, Denmark) weighing 200-240 g were used. When the rats were still on a standard diet (Altromin stock diet No. 1324, Chr. Petersen A/S, Ringsted, Denmark) blood was drawn to determine the starting levels of plasma lipids. The rats were then randomly divided into 2 experimental groups of 35 rats each, receiving casein diet without metoprolol (group C) or casein diet with 0.6% metoprolol (group Me), respectively. The pelleted diet containing casein protein (20%), olive oil (10%) cholesterol (0.5%) and cellulose powder (5.3%) was specially prepared by Ewos AB, Siidertalje, Sweden. The protein source was commercial casein containing 90% protein. The composition of vitamins and minerals was the same as described previously [lo]. The animals were housed in subgroups of 2, in macrolon cages (size 3) at 22°C with a 12 h (06.00-18.00 h) light-dark cycle. They were allowed free access to food and fresh tap water. Systolic blood pressure and heart rate were measured indirectly with the tail-cuff technique, as
previously described [ll]. During month 5 of the period of dietary treatment 3 determinations were performed and values were averaged. In addition, blood was drawn from the tail at defined times (1, 3 and 6 months) and used for plasma lipid analysis. After 9 months on the diet the rats were anesthetized with CO, and exsanguinated. Blood was collected in centrifuge tubes containing 40 pmoles EDTA, 1 nmole DTNB (5,5’-dithiobis(2nitrobenzoic acid)), inhibitor of the enzyme 1ecithin:cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) and 2000 KIE Trasylol@ (proteinase inhibitor, Bayer-Leverkusen) in 410 ~1 0.15 M sodium chloride. Plasma was separated by centrifugation at 500 X g for 20 min. The liver, kidneys, heart and aorta from each animal were collected and weighed. The heart was rinsed from residual blood with ice-cold 0.15 M saline. The heart and kidneys were fixed in 10% neutral buffered formalin and used for histopathological examination. Aortas were stored at - 20 o C until analysed. Very low density lipoproteins (VLDL) were isolated by preparative ultracentrifugation as described previously [12]. Agarose gel electrophoresis of lipoproteins was carried out at pH 7.0 as described elsewhere [13]. Plasma and VLDL fractions were analysed for cholesterol and triacylglycerols by enzymatic methods using kits supplied by Boehringer, Mannheim (Cat. Nos. 237 574, 310 328 and 126 012). High density lipoprotein-(HDL,)-cholesterol was measured after precipitation of other lipoproteins with dextran sulphate and magnesium chloride [14]. The plasma concentrations of metoprolol and one of its metabolites, hydroxy-metoprolol, were determined by high-resolution gas chromatography and mass spectrometric detection [15,16]. Aorta analysis Ten rats were randomly selected from each group for lipid and protein analysis of the aortas as described previously [17]. Morphological methods Since 4 rats of the control group and 1 rat of the metoprolol-treated group died during the study, the morphological investigation included 65
137 hearts and 43 aortas obtained from a metoprololtreated group and a control group. The metoprolol-treated group included 34 hearts and 24 aortas, while the control group consisted of 31 hearts and 19 aortas. The aortas were fixed in 10% formalin, frozen and cut on a cryostat at the following 5 levels: the aortic curve close to the heart, 3 sites of the thoracic and abdominal aorta and the inguinal aorta close to the bifurcation. For demonstration of intimal changes with and without lipid deposits, 15 sections (3 at each level) were stained with Oil Red 0. The distribution of aortic intimal plaques with and without lipids was demonstrated by light microscopy. All hearts were fixed in 10% formalin, sectioned, stained and evaluated as described previously [17].
TABLE
Antioxidants In order to protect lipids from oxidative damage, butylated hydroxyanisole (BHA) at a concentration of 0.005% (w/v) was added to blood plasma and to all organic solvent systems used.
Heart rate, blood pressure and growth Metoprolol treatment results in a significant decrease in heart rate (P < 0.002), as well as in blood pressure (P < 0.001) measured after 5 months on the diet (Table 1). A reduction in growth was observed in the metoprolol-treated group (Fig. l), whereas no effect on the weight of the heart, liver and kidneys/g body weight could be seen compared to the control group (Table 1).
Statistical methods Data were evaluated statistically and presented as means k standard deviations. Statistical analysis was performed by the Student t-test [18] at each time point since the variances at the different time points were assumed to be unequal. To compensate for the multiple testing the P-values were adjusted using the Bonferroni inequality. A difference was significant if P < 0.01. The frequency of hearts with sclerotic arteries with and without lipid deposits was analyzed with Fisher’s exact test
EFFECT OF METOPROLOL ON WEIGHT LIVER AND KIDNEYS AND ON HEART BLOOD PRESSURE
OF HEART. RATE AND
Mean + SD. Heart (g/100
g b.w.)
c (n = 31) Me (n = 34)
0.39f 0.395
0.02 0.04 1 ns
Liver (g/100
g b.w.)
c (n = 31) Me (n = 34)
5.16+ 5.23k
0.33 0.33 > ns
Kidneys (g/100 g b.w.)
C(n=31) Me (n = 34)
0.74+ 0.72k
0.05 0.04 > ”
Heart rate (beats/min)
c (n = 25) Me(n=29)
417 383
+36 *28 >
P < 0.002
Blood pressure
C (n = 25) Me(n=29)
160 139
+21 +I1 )
P < 0.001
(mm Hg) C = control
rats; Me = metoprolol-treated
rats.
Non-fasting plasma lipids Feeding an atherogenic diet (0.5% cholesterol and 20% casein protein) to SHR for 9 months resulted in an increase in non-fasting plasma lipids,
n91. Results
1
450 -
F
400r
H
Drug level in plasma The plasma level of metoprolol was determined at 1, 3, 6 and 9 months of metoprolol treatment and the mean plasma level of metoprolol during the investigation was calculated to be 92 k 64 nM. After 6 and 9 months of metoprolol treatment the major metabolite of metoprolol in rats retaining some blocking activity [16], hydroxy-metoprolol, was determined, resulting in a mean plasma level of 2174 + 1147 nM.
Y
ii
350-
300.
250 //,/ 1 0
5
10
15
20
25
30
35
40
TIME (weeks)
Fig. 1. Growth curves of male spontaneously hypertensive fed casein, 0.5% cholesterol (n = 31) (0) or casein, cholesterol, metoprolol (n = 34) (0). Mean f S.D.
rats 0.5%
138 TABLE
2
PLASMA
LIPIDS
OF SPONTANEOUSLY
HYPERTENSIVE
RATS FED CHOLESTEROL
ENRICHED
DIET
Mean f SD. Time
Months
Cholesterol Triglycerides
(mM) (mM)
Phosphohpids HDL,-cholesterol Number
(mM) (mM)
of rats
C Me C Me C Me C Me C Me
2.48 f 0.32 d 2.79 + 0.26 1 1.9lkO.47 2.36 +0.72 1 ’ 2.1OkO.23 2.47 f 0.21 e
35 35
35 35
C = control rats; Me = metoprolol-treated rats. a P > 0.05, b P < 0.05, = P < 0.01, d P < 0.001, e P < 0.0001. Note: a difference was declared significant if P < 0.01, because
3
LIPIDS
IN AORTA
_
33 34
32 34
of the multiple
cholesterol, triglycerides and phospholipids, 1608, 260% and 50%, respectively (Table 2). After the first month on the atherogenic diet the plasma levels of cholesterol, triglycerides and phospholipids of the metoprolol-treated rats were significantly increased compared to untreated control rats on the same diet. A significantly higher concentration of plasma triglycerides was also observed in the treated animals at 6 months, but no such difference in triglyceride concentration could be seen after 9 months on the diet. However, at the end of the investigation, the metoprolol-treated rats had a significantly lower
TABLE
_
testing
0.68 +0.09 0.70*0.11 31 34
situation.
plasma level of cholesterol and phospholipids, whereas no such effect could be demonstrated for the HDL,-cholesterol level (Table 2).
Aorta lipids The total content of lipids in the aorta was significantly higher in the metoprolol-treated rats than in control rats on the same atherogenic diet (Table 3). There was no difference in the cholesterol content but a significantly higher content of triglycerides in the aortas of metoprololtreated rats. The triglyceride concentrations of the
Mean + SD.
Total lipids (mg/mg protein) Cholesterol (pg/mg protein) Phospholipids ( pg/mg protein) Triglycerides (cg/mg protein) ’ Control rats on regular diet (Altromin b P > 0.05; c P < 0.05; d P < 0.0001.
Control rats (regular diet) a (n = 5)
Control rats (atherogenic diet) (n =lO)
Metoprolol-treated (atherogenic diet) (n =lO)
0.12* 0.03 5.4 f 0.3 12.6 + 2.3 90 zt25
0.10* 0.02 5.1 f 0.7 12.4 f 2.3 46 *15
0.13 4.6 10.1 87
stock diet, Chr. Petersen
A/S,
Ringsted,
Denmark)
[17].
* 0.02 = f 0.6 b f 2.6 ’ *22d
rats
139 TABLE
4
MYOCARDIAL AMETER z B
60
s w
40
e
20 0
8
80
g 5
60
s
40
iti II. 20
SCLEROTIC
ARTERIES
z 100 pm IN DI-
Control rats (n=31)
Metoprololtreated rats (n=34)
P ’
Frequency of hearts with sclerotic arteries (with and without lipid deposits) a
13/31
8/34
0.187
Frequency of hearts with sclerotic arteries with lipid deposits b
11/31
l/34
0.002
0 012345 NUMBER WITH
OF LIPID
SECTIONS
artery
of each heart: of each heart:
54. 3.
DEPOSITS
Fig. 2. Distribution of rats according to frequency of aortic intimal lipid deposits. The upper histogram represents the control group (n = 19) on the atherogenic diet, while the lower histogram represents the metoprolol-treated group (n = 24).
Fig. 3. Small intramyocardial
a Number of sections b Number of sections ’ Fisher’s test.
showing thickening
aortas of metoprolol-treated rats those found in rats fed a regular
normal structure in SHR (left) and small intramyocardial with lipid deposits in SHR (right). Oil Red 0, x 360.
artery
are similar diet (Table
showing
focal intimal
140 Cardiovascular pathology After 9 months on the atherogenic diet, 6 of the 19 control rats examined showed aortic intimal thickening containing accumulations of lipids, whereas only 2 of 24 metoprolol-treated rats showed similar changes. The lipid-containing plaques were located in the thoracic aorta. The frequency of aortic sections observed to contain intimal plaques in the different groups is given in Fig. 2. Intimal changes were also observed in the intramural coronary arteries (diameter > 100 pm). These changes consisted of components from proliferating cells and subendothelial deposits of a structureless material with and without lipid deposits (Fig. 3). Taking the total number of intimal changes into account, no significant difference could be demonstrated between the two groups (Table 4). However, a variation in the number of detected intramural arteries with intimal changes without (Fig. 4A) or with lipid accumulation (Fig. 4B) was observed. Intimal changes containing lipids were observed in 11 of 31 control rats fed an atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-
1001
0 0123456789 NUMBER OF SCLEROTIC ARTERIES
NUMBER OF ARTERIES WITH LIPID DEPOSITS
Fig. 4. (A) Distribution of rats according to number of detected intimal changes of intramural arteries of the heart. (B) Distribution of rats according to number of detected intimal changes with lipid deposits in the intramural arteries of the heart. The upper histograms represent control rats fed an atherogenic diet (n = 31), while the lower histograms represent metoprolol-treated rats (n = 34).
treated rats fed an otherwise 0.002) (Table 4).
identical
diet (p <
Discussion The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in SHR and to reveal any influence of treatment with metoprolol on these parameters. The occurrence of lesions was estimated by measuring both the lipid content of the aorta and the frequency of intimal lipid deposits in aorta and coronary arteries and the frequency of different types of intimal changes in the coronary arteries. The SHR strain, derived from Kyoto-Wistar [20], shows a more moderate plasma lipid response to diet than suspected on the grounds of studies on rats of the Sprague-Dawley strain [10,17]. Further, strain differences have recently been reported on the effect of dietary oils on serum lipid profiles, indicating that the Sprague-Dawley rat is more susceptible to diet-induced lipid modifications than the SHR [21]. Since the plasma cholesterol levels obtained in the present study were 4-5 mM only, one would not expect severe atherosclerotic changes. Therefore, the measurement of lipid accumulation of the total aorta was too insensitive a method to determine the small intimal lipid accumulation obtained in this study. Consequently, the cholesterol content of the aortas from rats on the atherogenic diet did not differ from rats on the regular diet [17]. Therefore, histopathological examination of the aorta and coronary arteries seems to be a more promising approach to detecting early intimal lipid deposits in rats. It has previously been reported that SHR on a regular diet show intimal and medial thickening of arteries. However, no lipid deposits in the intima have been described [22,23]. In this study, the rats obtained a diet that increased the plasma level of cholesterol during the investigation and caused detectable intimal lipid deposits in small arteries of the heart, as well as in the aorta. Metoprolol treatment caused a significant decrease in intimal lipid deposits in the small arteries of the heart, and possibly (although not statistically significant in the present study) in the aorta. Both biochem-
141 ical and hemodynamic factors have been suggested to be due to the antiatherogenic effects of P-blockers (24-28). In the present study, metopro101 treatment caused a decrease in heart rate and in blood pressure. At the end of the investigation a lower plasma level of cholesterol observed in the metoprolol-treated group could also contribute to the outcome of this study. However, an antiatherogenic effect of lipophilic /3-blockers such as propranolol [l-3] and metoprolol [4,5] has been directly demonstrated in cholesterol-fed rabbits, an effect which was not associated with a reduced level of serum cholesterol. As atherosclerosis is a multifactorial disease, there are several parameters not measured in this study that could be affected by fi-blockers [25-281. Whether there is some common mechanism behind the reduced growth rate and the reduced arterial lipid deposition due to metoprolol treatment, or some other causal connection between these two effects, cannot be decided from available data. In conclusion, lesions involving deposition of lipid in the arterial wall have been shown to occur in male SHR fed on an atherogenic diet for a 9-month period. A considerably longer experimental period is needed to decide whether these lesions are indeed related to the development of atherosclerosis. However, since metoprolol has been shown in recent studies to have an antiatherosclerotic effect in cholesterol-fed rabbits [4,5], it seems to provide protection against the present lipid-containing type of lesion in rats. Acknowledgements The authors wish to thank Ms. Angela Menschik and Ms. Ann-Sofie Lindh for excellent technical assistance. References Whittington-Coleman, P.J.. Carrier, Jr., 0. and Douglas, B.H., The effects of propranolol on diet-induced atheromatous lesions, Atherosclerosis, 18 (1973) 337. Spence, J.D.. Perkins, D.G., Kline, R.L., Adams, M.A. and Haust, M.D., Hemodynamic modification of aortic atherosclerosis. Effects of propranolol vs. hydralazine in hypertensive hyperlipidemic rabbits. Atherosclerosis, 50 (1984) 325.
A.V., Brecher, P. and Chan. C., Effects of 3 Chobanian, propranolol on atherogenesis in cholesterol-fed rabbits, Circ. Res., 56 (1985) 755. 4 ijstlund-Lindqvist. A.-M., Lindqvist, P.. Brlutigam, J., Olsson, G.. Bondjers. G. and Nordborg, C., Effect of metoprolol on diet-induced atherosclerosis in rabbits. Arteriosclerosis. 8 (1988) 40. 5 Lindqvist. P.. Olsson, G., Nordborg, C.. Bondjers. G.. Brlutigam, J. and &tlund-Lindqvist, A.-M.. Atherosclerosis in rabbits identified as high and low responders to an atherogenic diet and the effect of treatment with a PIblocker, Atherosclerosis, 72 (1988) 163. 6 Fairweather. F.A., Cardiovascular disease in rats. In: E. Cotchin and F.J.C. Roe (Eds.), Pathology of Laboratory Rats and Mice, Blackwell Scientific Publications, Oxford and Edinburgh. 1967. p. 213. 7 Russel. J.C. and Amy, R.M., Early atherosclerotic lesions in a susceptible rat model. The LA/N-corpulent rat. Atherosclerosis, 60 (1986) 119. 8 Peric-Golia. L. and Peric-Golia. M.. Aortic and renal lesions in hypercholesterolemic adult, male, virgin SpragueDawley rats, Atherosclerosis, 46 (1983) 57. 9 Yamori. Y., Nara. Y., Tagami. M., Mano. M., Kihara, M., Fujiwara, K. Horie, R. and Kamino, K., Common cellular disposition to hypertension and atherosclerosis. J. Hypertension. 2 (Suppl. 3) (1984). 10 Eklund, A. and Sjiiblom, L.. Effect of dietary proteins on hepatic and plasma lipids, and some properties of major plasma lipoprotein fractions during dietary-induced hypercholesterolemia in male rats. Biochim. Biophys. Acta, 877 (1986) 127. 11 Friberg, P., Folkow, B. and Nordlander, M., Cardiac dimensions in spontaneously hypertensive rats following different modes of blood pressure reduction by antihypertensive treatment, J. Hypertension. 4 (1986) 85. 12 Eklund, A., Sjoblom, L.. Gstlund-Lindqvist, A.-M.. Accumulation of cholesteryl esters and triglycerides in cultured macrophages incubated with plasma very low density lipoproteins from rats fed on casein or soybean protein diets containing moderate levels of cholesterol. Atherosclerosis. 62 (1986) 129. 13 Eklund, A. and Sjoblom. L., Improved banding pattern of rat plasma lipoproteins developed by agarose gel electrophoresis at pH 7.0, Biochim. Biophys. Acta. 877 (1986) 135. 14 Sjoblom, L. and Eklund. A., Determination of HDL,cholesterol by precipitation with dextran sulfate and magnesium chloride: Establishing optimal conditions for rat plasma, Lipids, 24 (1989) 532. 15 Ervik, M., Kylberg-Hanssen, K., Johansson, L., Determination of metoprolol in plasma and mine using high-resolution gas chromatography and electron-capture detection, J. Chromatography, 381 (1986) 168. 16 Borg. K.O.. Carlsson, E., Hoffmann. K.-J., Jonsson, T.-E., Thorin. H. and Wallin. B., Metabolism of metoprolol-( ‘H) in man, the dog and the rat, Acta Pharmacol. Toxicol.. 36 (1975) (Suppl. V) 125. 17 Sjoblom, L., Eklund, A., Gstlund-Lindqvist. A.-M. and Jiinsson. K.. Effects of diet and metoprolol on lipid levels
142
18
19 20 21
22
23
in the blood plasma and morphology of the heart and intramural branches of coronary arteries of spontaneously hypertensive male rats - A 9-month study, Ann. Nutr. Metab., (1989) in press. Snedecor, G.W. and Cochran, W.G., Statistical Methods, 6th edn., Iowa State University Press, Ames, IA, 1967, p. 17. Fleiss, J.L., Statistical Methods for Rates and Proportions, Wiley and Sons, New York, NY, 1981. Okamoto, K. and Aoki, K., Development of spontaneously hypertensive rats, Jpn. Circ. J., 27 (1963) 282. Kingsley, T.R. and Snyder, D.L., Serum lipids in spontaneously hypertensive rats and Sprague-Dawley rats fed menhaden oil, Lipids, 23 (1988) 564. Bucher, B., Travo, P. and Stoclet, J.-C., Smooth muscle cell hypertrophy and hyperplasia in the thoracic aorta of spontaneously hypertensive rats, Cell. Biol. Int. Reports, 8 (1984) 567. Hadjiisky, P., Peyri, N. and Grosgogeat, Y., Tunica media changes in the spontaneously hypertensive rat (SHR), Atherosclerosis, 65 (1987) 125.
24 Ablad, B., BjBrkman, J.-A., Gustafsson, D., Hansson, G., astlund-Lindqvist, A.-M. and Pettersson, K., The role of sympathetic activity in atherogenesis: effects of P-blockade, Am. Heart J., 116 (1988) 322. 25 Kaplan, J.R., Manuck, S.B., Adams, M.R., Weingaud, K.W. and Clarkson, T.B., The effect of beta-adrenergic blocking agents on atherosclerosis and its complications, Eur. Heart J., 8 (1987) 928. 26 Kaplan, J.R., Manuck, S.B., Adams, M.R., Weingaud, K.W. and Clarkson, T.B., Propranolol inhibits coronary atherosclerosis in behaviorally predisposed monkeys fed an atherogenic diet, Circulation, 76 (1987) 1364. 27 Pettersson, K. and Ablad, B., Metoprolol inhibits platelet deposition at arterial bifurcations in rabbits with sympathetic activation, FASEB J., 2 (1988) 7456. 28 Bell, F.P., Effects of antihypertensive agents propranolol, metoprolol, nadolol, prazosin, and chlorthalidone on ACAT activity in rabbit and rat aortas and on LCAT activity in human plasma in vitro, J. Cardiovasc. Pharmacol., 7 (1985) 437.
ATHERO
135 Ltd.
04408
Effect of metoprolol on plasma lipids and arterial intimal lipid deposition in spontaneously hypertensive rats A.-M. hstlund-Lindqvist
I, A. Eklund
*, L. Sjablom
* and L. Jijnsson
3
’Departmeni of Biochemistry, Hksle Research Laboratories, Miilndal, ’ Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala and ’ Department of Pathology, Swedish University of Agricultural Sciences, Uppsala (Sweden) (Received 25 January, 1989) (Revised, received 21 August, 1989) (Accepted 24 August, 1989)
Summary The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in spontaneously hypertensive rats (SHR) and to reveal any influence of treatment with metoprolol on these parameters. Metoprolol treatment caused an 8% decrease in heart rate and a 13% decrease in blood pressure and led to a rise in plasma triglycerides, 24%, 17% and 34% after 1, 3 and 6 months of metoprolol treatment, respectively. However, no effect on plasma triglycerides was observed after 9 months of metoprolol treatment while a reduced cholesterolemic response was observed. Intimal proliferations containing accumulations of lipids were observed in small intramural branches of coronary arteries (> 100 pm) in 11 of 31 control rats fed the atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-treated rats fed an otherwise identical diet. The corresponding figures for the frequency of lipid containing intimal plaques in aorta were 6/19 in controls and 2/24 in the metoprolol-treated group.
Key words:
Atherosclerosis; Metoprolol
Aorta;
Coronary
arteries;
Introduction In previous studies, non-selective P-blockers have been shown to have an anti-atherogenic ef-
Correspondence to: A.-M. ijstlund-Lindqvist, Ph.D., Department of Biochemistry, HBssle Research Laboratories, S-431 83 Miilndal, Sweden. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
Lipids;
Spontaneously
hypertensive
rats;
feet in cholesterol-fed rabbits [l-3]. Recently, the same effect has been demonstrated for the adrenergic &-receptor antagonist, metoprolol [4,5]. The mechanism underlying this effect is not known. In order to test the species-specificity of the phenomenon, we wanted to extend the studies of the anti-atherogenic effect of metoprolol by looking for possible influences of metoprolol in a different animal species. Ltd.
136 In contrast to the rabbit, which has been the most frequently used experimental animal in atherosclerosis research, the rat has been considered very resistant to atherosclerosis. However, rats develop atherosclerosis as they grow old [6] and certain strains of rat, such as the LA/N corpulent rat [7], develop atherosclerosis within about 9 months. Sclerotic lesions, consisting of raised intimal plaques in the aorta, have also been found in all male Sprague-Dawley-strain rats reaching a plasma cholesterol level above 3.9 mmol/l in a study extending over 80 weeks [8]. Furthermore, the stroke-prone spontaneously hypertensive rats (SHRSP) [9] rapidly develop arterial fat deposits when fed on high cholesterol diets. As both hypertension and hyperlipidemia are well-known risk factors for the development of atherosclerosis, we decided to test the effect of metoprolol treatment on atherosclerosis and on plasma levels of lipids in spontaneously hypertensive male rats (SHR) fed an atherogenic diet. Materials and methods Experimental design Seventy male, inbred, spontaneously hypertensive SHR/ Molrats (Mollegaard, Ltd., Skensved, Denmark) weighing 200-240 g were used. When the rats were still on a standard diet (Altromin stock diet No. 1324, Chr. Petersen A/S, Ringsted, Denmark) blood was drawn to determine the starting levels of plasma lipids. The rats were then randomly divided into 2 experimental groups of 35 rats each, receiving casein diet without metoprolol (group C) or casein diet with 0.6% metoprolol (group Me), respectively. The pelleted diet containing casein protein (20%), olive oil (10%) cholesterol (0.5%) and cellulose powder (5.3%) was specially prepared by Ewos AB, Siidertalje, Sweden. The protein source was commercial casein containing 90% protein. The composition of vitamins and minerals was the same as described previously [lo]. The animals were housed in subgroups of 2, in macrolon cages (size 3) at 22°C with a 12 h (06.00-18.00 h) light-dark cycle. They were allowed free access to food and fresh tap water. Systolic blood pressure and heart rate were measured indirectly with the tail-cuff technique, as
previously described [ll]. During month 5 of the period of dietary treatment 3 determinations were performed and values were averaged. In addition, blood was drawn from the tail at defined times (1, 3 and 6 months) and used for plasma lipid analysis. After 9 months on the diet the rats were anesthetized with CO, and exsanguinated. Blood was collected in centrifuge tubes containing 40 pmoles EDTA, 1 nmole DTNB (5,5’-dithiobis(2nitrobenzoic acid)), inhibitor of the enzyme 1ecithin:cholesterol acyltransferase (LCAT, E.C. 2.3.1.43) and 2000 KIE Trasylol@ (proteinase inhibitor, Bayer-Leverkusen) in 410 ~1 0.15 M sodium chloride. Plasma was separated by centrifugation at 500 X g for 20 min. The liver, kidneys, heart and aorta from each animal were collected and weighed. The heart was rinsed from residual blood with ice-cold 0.15 M saline. The heart and kidneys were fixed in 10% neutral buffered formalin and used for histopathological examination. Aortas were stored at - 20 o C until analysed. Very low density lipoproteins (VLDL) were isolated by preparative ultracentrifugation as described previously [12]. Agarose gel electrophoresis of lipoproteins was carried out at pH 7.0 as described elsewhere [13]. Plasma and VLDL fractions were analysed for cholesterol and triacylglycerols by enzymatic methods using kits supplied by Boehringer, Mannheim (Cat. Nos. 237 574, 310 328 and 126 012). High density lipoprotein-(HDL,)-cholesterol was measured after precipitation of other lipoproteins with dextran sulphate and magnesium chloride [14]. The plasma concentrations of metoprolol and one of its metabolites, hydroxy-metoprolol, were determined by high-resolution gas chromatography and mass spectrometric detection [15,16]. Aorta analysis Ten rats were randomly selected from each group for lipid and protein analysis of the aortas as described previously [17]. Morphological methods Since 4 rats of the control group and 1 rat of the metoprolol-treated group died during the study, the morphological investigation included 65
137 hearts and 43 aortas obtained from a metoprololtreated group and a control group. The metoprolol-treated group included 34 hearts and 24 aortas, while the control group consisted of 31 hearts and 19 aortas. The aortas were fixed in 10% formalin, frozen and cut on a cryostat at the following 5 levels: the aortic curve close to the heart, 3 sites of the thoracic and abdominal aorta and the inguinal aorta close to the bifurcation. For demonstration of intimal changes with and without lipid deposits, 15 sections (3 at each level) were stained with Oil Red 0. The distribution of aortic intimal plaques with and without lipids was demonstrated by light microscopy. All hearts were fixed in 10% formalin, sectioned, stained and evaluated as described previously [17].
TABLE
Antioxidants In order to protect lipids from oxidative damage, butylated hydroxyanisole (BHA) at a concentration of 0.005% (w/v) was added to blood plasma and to all organic solvent systems used.
Heart rate, blood pressure and growth Metoprolol treatment results in a significant decrease in heart rate (P < 0.002), as well as in blood pressure (P < 0.001) measured after 5 months on the diet (Table 1). A reduction in growth was observed in the metoprolol-treated group (Fig. l), whereas no effect on the weight of the heart, liver and kidneys/g body weight could be seen compared to the control group (Table 1).
Statistical methods Data were evaluated statistically and presented as means k standard deviations. Statistical analysis was performed by the Student t-test [18] at each time point since the variances at the different time points were assumed to be unequal. To compensate for the multiple testing the P-values were adjusted using the Bonferroni inequality. A difference was significant if P < 0.01. The frequency of hearts with sclerotic arteries with and without lipid deposits was analyzed with Fisher’s exact test
EFFECT OF METOPROLOL ON WEIGHT LIVER AND KIDNEYS AND ON HEART BLOOD PRESSURE
OF HEART. RATE AND
Mean + SD. Heart (g/100
g b.w.)
c (n = 31) Me (n = 34)
0.39f 0.395
0.02 0.04 1 ns
Liver (g/100
g b.w.)
c (n = 31) Me (n = 34)
5.16+ 5.23k
0.33 0.33 > ns
Kidneys (g/100 g b.w.)
C(n=31) Me (n = 34)
0.74+ 0.72k
0.05 0.04 > ”
Heart rate (beats/min)
c (n = 25) Me(n=29)
417 383
+36 *28 >
P < 0.002
Blood pressure
C (n = 25) Me(n=29)
160 139
+21 +I1 )
P < 0.001
(mm Hg) C = control
rats; Me = metoprolol-treated
rats.
Non-fasting plasma lipids Feeding an atherogenic diet (0.5% cholesterol and 20% casein protein) to SHR for 9 months resulted in an increase in non-fasting plasma lipids,
n91. Results
1
450 -
F
400r
H
Drug level in plasma The plasma level of metoprolol was determined at 1, 3, 6 and 9 months of metoprolol treatment and the mean plasma level of metoprolol during the investigation was calculated to be 92 k 64 nM. After 6 and 9 months of metoprolol treatment the major metabolite of metoprolol in rats retaining some blocking activity [16], hydroxy-metoprolol, was determined, resulting in a mean plasma level of 2174 + 1147 nM.
Y
ii
350-
300.
250 //,/ 1 0
5
10
15
20
25
30
35
40
TIME (weeks)
Fig. 1. Growth curves of male spontaneously hypertensive fed casein, 0.5% cholesterol (n = 31) (0) or casein, cholesterol, metoprolol (n = 34) (0). Mean f S.D.
rats 0.5%
138 TABLE
2
PLASMA
LIPIDS
OF SPONTANEOUSLY
HYPERTENSIVE
RATS FED CHOLESTEROL
ENRICHED
DIET
Mean f SD. Time
Months
Cholesterol Triglycerides
(mM) (mM)
Phosphohpids HDL,-cholesterol Number
(mM) (mM)
of rats
C Me C Me C Me C Me C Me
2.48 f 0.32 d 2.79 + 0.26 1 1.9lkO.47 2.36 +0.72 1 ’ 2.1OkO.23 2.47 f 0.21 e
35 35
35 35
C = control rats; Me = metoprolol-treated rats. a P > 0.05, b P < 0.05, = P < 0.01, d P < 0.001, e P < 0.0001. Note: a difference was declared significant if P < 0.01, because
3
LIPIDS
IN AORTA
_
33 34
32 34
of the multiple
cholesterol, triglycerides and phospholipids, 1608, 260% and 50%, respectively (Table 2). After the first month on the atherogenic diet the plasma levels of cholesterol, triglycerides and phospholipids of the metoprolol-treated rats were significantly increased compared to untreated control rats on the same diet. A significantly higher concentration of plasma triglycerides was also observed in the treated animals at 6 months, but no such difference in triglyceride concentration could be seen after 9 months on the diet. However, at the end of the investigation, the metoprolol-treated rats had a significantly lower
TABLE
_
testing
0.68 +0.09 0.70*0.11 31 34
situation.
plasma level of cholesterol and phospholipids, whereas no such effect could be demonstrated for the HDL,-cholesterol level (Table 2).
Aorta lipids The total content of lipids in the aorta was significantly higher in the metoprolol-treated rats than in control rats on the same atherogenic diet (Table 3). There was no difference in the cholesterol content but a significantly higher content of triglycerides in the aortas of metoprololtreated rats. The triglyceride concentrations of the
Mean + SD.
Total lipids (mg/mg protein) Cholesterol (pg/mg protein) Phospholipids ( pg/mg protein) Triglycerides (cg/mg protein) ’ Control rats on regular diet (Altromin b P > 0.05; c P < 0.05; d P < 0.0001.
Control rats (regular diet) a (n = 5)
Control rats (atherogenic diet) (n =lO)
Metoprolol-treated (atherogenic diet) (n =lO)
0.12* 0.03 5.4 f 0.3 12.6 + 2.3 90 zt25
0.10* 0.02 5.1 f 0.7 12.4 f 2.3 46 *15
0.13 4.6 10.1 87
stock diet, Chr. Petersen
A/S,
Ringsted,
Denmark)
[17].
* 0.02 = f 0.6 b f 2.6 ’ *22d
rats
139 TABLE
4
MYOCARDIAL AMETER z B
60
s w
40
e
20 0
8
80
g 5
60
s
40
iti II. 20
SCLEROTIC
ARTERIES
z 100 pm IN DI-
Control rats (n=31)
Metoprololtreated rats (n=34)
P ’
Frequency of hearts with sclerotic arteries (with and without lipid deposits) a
13/31
8/34
0.187
Frequency of hearts with sclerotic arteries with lipid deposits b
11/31
l/34
0.002
0 012345 NUMBER WITH
OF LIPID
SECTIONS
artery
of each heart: of each heart:
54. 3.
DEPOSITS
Fig. 2. Distribution of rats according to frequency of aortic intimal lipid deposits. The upper histogram represents the control group (n = 19) on the atherogenic diet, while the lower histogram represents the metoprolol-treated group (n = 24).
Fig. 3. Small intramyocardial
a Number of sections b Number of sections ’ Fisher’s test.
showing thickening
aortas of metoprolol-treated rats those found in rats fed a regular
normal structure in SHR (left) and small intramyocardial with lipid deposits in SHR (right). Oil Red 0, x 360.
artery
are similar diet (Table
showing
focal intimal
140 Cardiovascular pathology After 9 months on the atherogenic diet, 6 of the 19 control rats examined showed aortic intimal thickening containing accumulations of lipids, whereas only 2 of 24 metoprolol-treated rats showed similar changes. The lipid-containing plaques were located in the thoracic aorta. The frequency of aortic sections observed to contain intimal plaques in the different groups is given in Fig. 2. Intimal changes were also observed in the intramural coronary arteries (diameter > 100 pm). These changes consisted of components from proliferating cells and subendothelial deposits of a structureless material with and without lipid deposits (Fig. 3). Taking the total number of intimal changes into account, no significant difference could be demonstrated between the two groups (Table 4). However, a variation in the number of detected intramural arteries with intimal changes without (Fig. 4A) or with lipid accumulation (Fig. 4B) was observed. Intimal changes containing lipids were observed in 11 of 31 control rats fed an atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-
1001
0 0123456789 NUMBER OF SCLEROTIC ARTERIES
NUMBER OF ARTERIES WITH LIPID DEPOSITS
Fig. 4. (A) Distribution of rats according to number of detected intimal changes of intramural arteries of the heart. (B) Distribution of rats according to number of detected intimal changes with lipid deposits in the intramural arteries of the heart. The upper histograms represent control rats fed an atherogenic diet (n = 31), while the lower histograms represent metoprolol-treated rats (n = 34).
treated rats fed an otherwise 0.002) (Table 4).
identical
diet (p <
Discussion The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in SHR and to reveal any influence of treatment with metoprolol on these parameters. The occurrence of lesions was estimated by measuring both the lipid content of the aorta and the frequency of intimal lipid deposits in aorta and coronary arteries and the frequency of different types of intimal changes in the coronary arteries. The SHR strain, derived from Kyoto-Wistar [20], shows a more moderate plasma lipid response to diet than suspected on the grounds of studies on rats of the Sprague-Dawley strain [10,17]. Further, strain differences have recently been reported on the effect of dietary oils on serum lipid profiles, indicating that the Sprague-Dawley rat is more susceptible to diet-induced lipid modifications than the SHR [21]. Since the plasma cholesterol levels obtained in the present study were 4-5 mM only, one would not expect severe atherosclerotic changes. Therefore, the measurement of lipid accumulation of the total aorta was too insensitive a method to determine the small intimal lipid accumulation obtained in this study. Consequently, the cholesterol content of the aortas from rats on the atherogenic diet did not differ from rats on the regular diet [17]. Therefore, histopathological examination of the aorta and coronary arteries seems to be a more promising approach to detecting early intimal lipid deposits in rats. It has previously been reported that SHR on a regular diet show intimal and medial thickening of arteries. However, no lipid deposits in the intima have been described [22,23]. In this study, the rats obtained a diet that increased the plasma level of cholesterol during the investigation and caused detectable intimal lipid deposits in small arteries of the heart, as well as in the aorta. Metoprolol treatment caused a significant decrease in intimal lipid deposits in the small arteries of the heart, and possibly (although not statistically significant in the present study) in the aorta. Both biochem-
141 ical and hemodynamic factors have been suggested to be due to the antiatherogenic effects of P-blockers (24-28). In the present study, metopro101 treatment caused a decrease in heart rate and in blood pressure. At the end of the investigation a lower plasma level of cholesterol observed in the metoprolol-treated group could also contribute to the outcome of this study. However, an antiatherogenic effect of lipophilic /3-blockers such as propranolol [l-3] and metoprolol [4,5] has been directly demonstrated in cholesterol-fed rabbits, an effect which was not associated with a reduced level of serum cholesterol. As atherosclerosis is a multifactorial disease, there are several parameters not measured in this study that could be affected by fi-blockers [25-281. Whether there is some common mechanism behind the reduced growth rate and the reduced arterial lipid deposition due to metoprolol treatment, or some other causal connection between these two effects, cannot be decided from available data. In conclusion, lesions involving deposition of lipid in the arterial wall have been shown to occur in male SHR fed on an atherogenic diet for a 9-month period. A considerably longer experimental period is needed to decide whether these lesions are indeed related to the development of atherosclerosis. However, since metoprolol has been shown in recent studies to have an antiatherosclerotic effect in cholesterol-fed rabbits [4,5], it seems to provide protection against the present lipid-containing type of lesion in rats. Acknowledgements The authors wish to thank Ms. Angela Menschik and Ms. Ann-Sofie Lindh for excellent technical assistance. References Whittington-Coleman, P.J.. Carrier, Jr., 0. and Douglas, B.H., The effects of propranolol on diet-induced atheromatous lesions, Atherosclerosis, 18 (1973) 337. Spence, J.D.. Perkins, D.G., Kline, R.L., Adams, M.A. and Haust, M.D., Hemodynamic modification of aortic atherosclerosis. Effects of propranolol vs. hydralazine in hypertensive hyperlipidemic rabbits. Atherosclerosis, 50 (1984) 325.
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24 Ablad, B., BjBrkman, J.-A., Gustafsson, D., Hansson, G., astlund-Lindqvist, A.-M. and Pettersson, K., The role of sympathetic activity in atherogenesis: effects of P-blockade, Am. Heart J., 116 (1988) 322. 25 Kaplan, J.R., Manuck, S.B., Adams, M.R., Weingaud, K.W. and Clarkson, T.B., The effect of beta-adrenergic blocking agents on atherosclerosis and its complications, Eur. Heart J., 8 (1987) 928. 26 Kaplan, J.R., Manuck, S.B., Adams, M.R., Weingaud, K.W. and Clarkson, T.B., Propranolol inhibits coronary atherosclerosis in behaviorally predisposed monkeys fed an atherogenic diet, Circulation, 76 (1987) 1364. 27 Pettersson, K. and Ablad, B., Metoprolol inhibits platelet deposition at arterial bifurcations in rabbits with sympathetic activation, FASEB J., 2 (1988) 7456. 28 Bell, F.P., Effects of antihypertensive agents propranolol, metoprolol, nadolol, prazosin, and chlorthalidone on ACAT activity in rabbit and rat aortas and on LCAT activity in human plasma in vitro, J. Cardiovasc. Pharmacol., 7 (1985) 437.