Atherosclerosis, 95 (1992) 17-85 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/$05.00
Printed and Published in Ireland
ATHERO 04854
Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine Alexander N. Orekhov, Irina V. Andrianova, Marc D. Rekhter, Vladimir V. Tertov, Elena R. Andreeva, Sergey E. Ragimov and Alexander A. Mironov Institute of Experimental Cardiology, National Cardiology Research Center, 3rd Cherepkovskaya Street I5 A, 121552 Moscow (Russia)
(Received 27 January, 1992) (Revised, received 7 April, 1992) (Accepted 8 April, 1992)
Summary The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally refered to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate betablocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy. Key words: Atherosclerosis; Beta-blockers; Calcium antagonists; Cell culture; Foam cells; Macrophages; Rabbit aorta injury; Smooth muscle cells
Correspondence IO: Alexander N. Orekhov, Ph.D., Institute of Experimental Cardiology, National Cardiology Research Center, 3rd Cherepkovskaya Street 15 A, 121552 Moscow, Russia. Tel.: (095) 149-0559; Fax: (095) 415-2962, (095) 4146699.
Introduction We have recently reported that the betablockers propranolol, alprenolol, metoprolol,
78
atenolol, pindolol and timolol increase intracellular cholesterol content and stimulate proliferative activity of smooth muscle cells cultured from human aortic intima [ 1,2]. These data allowed us to suggest that beta-blockers elicit a direct atherogenic effect at the arterial cell level. Since intracellular lipid administration and stimulation of cell proliferation are the earliest manifestations of atherosclerosis at the cell level, the ability of betablockers to stimulate these processes in vitro was conventionally termed an atherogenic potential or [ 1,2]. Atherogenicity of proatherogenicity pranolol was also revealed when blood sera of patients were examined. Blood serum obtained after a peroral administration of the drug stimulated proliferation of cultured cells and induced accumulation of intracellular cholesterol [ 1,2]. The atherogenicity of beta-blockers elicited in a cell culture can be considerably reduced or eliminated by combining them with other drugs. For example, nitroglycerin and the calcium antagonist, nifedipine, abolished the stimulation of cell proliferation and intracellular accumulation of cholesterol induced by metoprolol [2]. The data mentioned were obtained on models in vitro (addition of the drug tested to cell culture) or ex vivo (addition of patient’s blood serum after drug administration to cell culture). A question arises whether the atherogenicity of beta-blockers occurs in vivo. If so, can this atherogenicity be eliminated by simultaneous administration of beta-blockers with other drugs? The answer to this question can be provided by in vivo investigations. In this study rabbits were used to find out whether the atherogenicity of beta-blockers is elicited after a peroral administration of these drugs. After the atherogenicity of beta-blockers had been revealed in vitro, ex vivo and in vivo, we attempted to eliminate the atherogenicity of the beta-blocker, propranolol, by combining it with papaverine. Materials and Methods
Propranolol (Anaprilin) and papaverine were purchased from Zdorov’e ChemicoPharmaceutical Corporation (Kharkov, USSR). Metoprolol (Spesicor) was from Laaketehdas Leiras Huhtamaki Oy (Turku, Finland), atenolol (Tenolol) from IPCA Laboratories Ltd. (Bombay,
India), pindolol (Visken) from EGIS (Budapest, Hungary) and timolol (Ocupres-E) from Cadila Laboratories Pvt. Ltd. (Ahmedabad, India). For in vitro experiments, alprenolol hydrochloride and metoprolol bitartrate were from Astra Pharmaceuticals Production AB (Sodertalje, Sweden); papaverine hydrochloride, propranolol hydrochloride, pindolol, atenolol, timolol maleate were purchased from Sigma Chemical Company (St. Louis, MO). Experiments were performed on male 12-15week Chinchilla rabbits weighing 3.0-3.5 kg. Animals were maintained on standard diet. Rabbit blood serum was added to cultured peritoneal macrophages of BALB/c mice obtained as described [3]. Cells were washed with Medium 199 and incubated for 4 h in medium 199 containing glutamine, antibiotics and 10% of the serum tested. Cells were extensively washed and the total cholesterol content was determined as previously 141. Human aortic subendothelial intimal smooth muscle cells were isolated from autopsy obtained within 2-3 h after death. Cells were harvested by enzymatic digestion of grossly normal part of aortic intima with 0.1% collagenase for 3-4 h and seeded into 24-well culture plates. Intimal cells were cultured for 7 days in Medium 199, containing 10% fetal calf serum, glutamine and antibiotics. On the seventh day of cultivation, test agents were added and total cholesterol content was determined in 24 h. Protocols for cell isolation and cultivation as well as technical details of the experiment are presented elsewhere [5]. Injury of rabbit aorta was produced under Nembutal anesthesia. The abdominal aorta was deendothelized with a 4F balloon catheter (61. After the operation, rabbits were assigned into 4 groups of 7-8 animals each. Group I animals received propranolol perorally at a dose of 20 mg, group II 20 mg papaverine, group III propranolol and papaverine and group IV 1 ml of distilled water. The drugs were dissolved in 1 ml of distilled water and given 3 times a day (at lO:OO,14:00 and 18:00 h) during a 21-day period. The whole experiment was repeated 3 times independently. The aorta was dissected on day 21 after deendothelization. A strip of aorta (5 mm wide) from the central part of the injury was fixed with 2.5%
79
glutaraldehyde for morphological examination. The thickness of media and intima was measured in semi-thin sections under a light microscope with the aid of a micrometer. The remainder of the aorta was used in biochemical studies. Neointima and media were separated mechanically, lipids were extracted with a chloroformmethanol mixture (2: 1) and separated by thin-layer chromatography. Phospholipids, free cholesterol, cholesterol esters and triglycerides ‘were quantitated by scanning densitometry [7]. The collagen content and cell number in the neointima were determined as described elsewhere [8]. The significance of differences was evaluated by dispersion analysis methods using a BMDP statistical program package 19). Results
Atherogenicity of beta-blockers In vitro. After 24 h of incubation with primary culture of human aortic intimal cells, the betablockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol induced a considerable increase in the total intracellular cholesterol content (Table 1). This confirms our previous observations [ 1,2]. Similar results were obtained in a culture of mouse peritoneal macrophages: after 4 h of incubation with beta-blockers intracellular cholesterol content increased by 30-90% (Table 1). Since the atherogenic effects of beta-blockers originally revealed in cultured human aortic cells are readily reproduced in cultured mouse peritoneal macrophages and taking into account that experiments on mouse macrophages are less laborious than those on human cells, in subsequent studies we employed a culture of mouse peritoneal macrophages. Ex vivo. Serum prepared from rabbit blood obtained 1,2,3,4 and 5 h after peroral administration of beta-blockers was added to a culture of peritoneal macrophages. Serum obtained 1 h after propranolol induced a substantial accumulation of cholesterol in the cells, i.e. exhibited atherogenicity (Fig. 1). Atherogenicity of the serum obtained 2 h after the drug administration was maximal and was retained during 4 h (Fig. 1). Similar effects were observed with blood sera obtained after peroral administration of metopro-
TABLE 1 CHOLESTEROL CONTENT WITH BETA-BLOCKERS
IN
CELLS
CULTURED
All drugs were tested in concentration of lo-’ M. Data are expressed as mean of 4 determinations f S.E.M. Cells were cultured in Medium 199, containing 10% fetal calf serum, glutamine and antibiotics as described in Materials and Methods. Control levels of total cholesterol in cultured cells were 53 * 4 pglmg cell protein (aortic cells) and 26 ?? 2 &mg cell protein (macrophages). n.a., not assayed. Drug
None (control) Propranolol Metoprolol Atenolol Pindolol Alprenolol Timolol
Cholesterol content (% of control) Human aortic intimal smooth muscle cells
Mouse peritoneal macrophages
loo* 158 zt 144 * 139 f 140 f 151 * 154 f
loo* 8 187 zt 11’ 182 ?? 10” 187 zt lO* n.a. 154 f 18* 132 f 9*
3 5’ 7* 9* 8’ 108 8*
*Significant difference from control, P < 0.05.
propranolol
Fig. 1. Effect of peroral administration of propranolol and propranolol-papaverine combination on the ability of rabbit serum to induce cholesterol accumulation in cultured macrophages. Propranolol (20 mg) or combination of propranolol (20 mg) plus papaverine (20 mg) were administered perorally. The time of administration is indicated with an arrow. Blood was collected 1,2, 3,4 and 5 h after administration. Blood serum obtained before (0 h) and after the drug administration were added to a culture of mouse peritoneal macrophages at a final concentration of 10%. The intracellular cholesterol content was determined 4 h afterwards. The cholesterol content in cells incubated with blood serum obtained before the drug administration was assumed as 100%. Each point represents the mean of 6-8 determinations in separate experiments.
80 TABLE 2 CHOLESTEROL CONTENT IN MOUSE PERITONEAL MACROPHAGES CULTURED WITH BLOOD SERUM OF RABBITS TREATED WITH BETA-BLOCKERS Data are expressed as mean of 4 determinations f S.E.M. Blood was drawn 2 h after oral drug administration and blood serum was added to cultured mouse peritoneal macrophages. Cell cholesterol content was measured 4 h later. Drug
Dose (mg)
Cholesterol content (“h of control)
None (control) Propranolol Metoprolol Atenolol Pindolol Timolol
-
100 195 f 126 * 161 zt 129 zt 194 f
20 50 100 5 2.5
5* 9* IO* 8* 19*
*Significant difference from control, P < 0.05.
101, atenolol, pindolol and timolol. These agents induced serum atherogenicity which manifested itself as cholesterol accumulation in cultured macrophages (Table 2). The beta-blocker propranolol was chosen for a detailed examination since it induced maximum atherogenicity both in vitro and ex vivo. Two hours after the first dose of propranolol(20 mg) rabbit blood serum became atherogenic,
i.e. it induced a 2.5fold accumulation of intracellular cholesterol (Fig. 2). Four hours after the first dose, blood atherogenicity was slightly decreased, remaining, however, at an essentially high level and it increased after the second dose (Fig. 2). By 4 h after the second dose (8 h after the first dose) serum atherogenicity decreased again but it was elevated after the third dose (Fig. 2). Twenty-four hours after the first dose followed by two doses given at 4-h intervals, serum atherogenicity remained high and increased a little after each subsequent dose (Fig. 2). Regular peroral administration of propranolol at 4-h intervals for 21 days maintained the atherogenicity of rabbit serum at a high level during the entire period (Fig. 3). In viva In intact rabbit aorta the intima was not pronounced (Fig. 4). Injury produced with a balloon catheter induced a considerable thickening of intima, i.e. formation of neointima (Fig. 4). Microscopically, 21 days after ballonization an elevation was detected in the injury zone in all aortas. On average, the thickness of neointima was one half of that of the media. The thickness of neointima in the aorta of control rabbits and rabbits given propranolol was compared in three independent experiments. Visually, the neointima of propranolol-treated rabbits was considerably thicker than that of control animals (Fig. 4). On
6
a
fg w_, 2 E 150-
Pz
Oal@ 3
0”
----------------------
=O- I 0
I
I 8
I
18
I 24
c
3;
HOURS Fig. 2. Effect of three peroral doses of propranolol on atherogenicity of rabbit blood serum. The atherogenicity of blood serum displayed as stimulation of cholesterol accumulation in cultured macrophages was evaluated as described in the legend to Fig. 1. The time of propranolol administration are indicated with arrows. Other details are the same as in the legends to Fig. 1.
0
5ot
I
0
I
10
I
20
DAYS Fig. 3. Effect of a long-term propranolol treatment on the atherogenicity of rabbit blood serum. Blood was collected on day 1, 2, 3, 7, 14 and 21 in the morning before and 2 h after the drug administration. Other details are the same as in the legends to Fig. 1.
82 TABLE 3 INFLUENCE OF PAPAVERINE ON PROPRANOLOL Parameter
Intima:media ratio Cell number cells/mg wet weight Cholesteryl esters &mg dry weight Free cholesterol pg/mg dry weight Triglycerides &mg dry weight Phospholipids pglmg dry weight Collagen &mg wet weight
Control values (intact rabbit aortas)
ATHEROGENICITY
% of control Control
Propranolol
Papaverine
0.48 + 0.01
loo
203 f 20*
96 f 8
94 f 8
(14.2 zt 1.2) x IO3
100
305 f 99
II6 f 4
127 zt 8
loo
573
107 ?? I1
94zt 16
27.7 zk 2.3
100
244 zt 32*
124 f I3
95 f IO
11.1 f 1.3
loo
355
?? 31*
161 + 38
158 f 26
21.9 zt 0.9
100
198 f 20.
n.a.
n.a.
331 f 26
100
172 f 20*
104 + 8
1.7
?? 0.4
?? 1358
Propranolol + papaverine
99*
IO
*Significant difference from control, P < 0.05. The data are calculated per intimal surface.
eliminated the atherogenicity of propranolol in vitro. Ex viva Simultaneous peroral administration of propranolol (20 mg) and papaverine (20 mg and more) prevented the appearance of atherogenic properties of rabbit blood serum (Fig. 1). Papaverine at a dose of 10 mg and lower did not eliminate the atherogenicity of propranolol completely but retarded its manifestations (Table 5). Regular administration of the propranololTABLE 4 CHOLESTEROL CONTENT IN MOUSE PERITONEAL MACROPHAGES CULTURED WITH PROPRANOLOL AND PAPAVERINE Details are the same as in Table 1 Drug
Cholesterol accumulation (% of control)
None (control) Propranolol Papaverine Propranolol + papaverine
100 zt 187 zt 102 f 93 zt
8 Ii+ I2 8
?? Significant difference from control, P < 0.05,
papaverine combination during 21 days resulted in a complete elimination of the propranolol atherogenicity (Fig. 3). Thus, papaverine abolished the atherogenic effect of propranolol applied in a single dose and in a long-term treatment. It should be mentioned that propranolol, papaverin, or propranolol-papaverin combination did not produce any effect on the blood cholesterol content during a 21-day period (not illustrated). In viva. Aortic neointima of rabbits given propranolol alone was considerably thicker than that of rabbits given propranolol with papaverine. Visually, the latter was similar to the neointima of control animals (Fig. 4). Accurate measurements confirmed the visual observations: on average the thickness of the neointima of rabbits given propranolol-papaverine was 2-fold less than that of propranolol-treated rabbits and equal to that of controls (Table 3). Papaverine alone had no effect on the intima thickness (Table 3). Papaverine completely prevented the propranolol-induced increase in the cell number and in lipid and collagen contents (Table 3). Papaverine by itself produced no effect on these indices (Table 3).
83 TABLE 5 EFFECT OF PROPRANOLOL AND PROPRANOLOL-PAPAVERINE CULTURED MACROPHAGES
COMBINATION ON CHOLESTEROL CONTENT IN
Blood serum obtained before (0 h) and after peroral drug administration was added to a culture of mouse peritoneal macrophages at a final concentration of 10%. The intracellular cholesterol content was determined 4 h afterwards. The cholesterol content in cells incubated with blood serum obtained before the drug administration was assumed as 100%. Other details are the same as in Table 2. Hours after administration
Administration
Propranolol, 20 Propranolol, 20 + papaverine, Propranolol, 20 + papaverine,
mg mg 10 mg mg 20 mg
0
1
2
3
4
100 ?? 10 loo* 5
145 zt 18 121 f 10
190 f 19* 134 f 14
176 zt 18; 174 f 14*
135 ?? 15’ 140 + 5*
loo*
105 ?? 17
loo
100 ?? 11
5
Discussion of mouse peritoneal Using a culture macrophages, we have confirmed our previous findings that beta-blockers are atherogenic at the cellular level [ 1,2], All beta-blockers examined (propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol) induced cholesterol accumulation in cultured cells. Peroral administration of these beta-blockers generated an atherogenie potential in rabbit blood serum, i.e., they acquired the ability to induce intracellular cholesterol accumulation. Long-term propranolol treatment produced a sustained blood atherogenicity The atherogenic potential of blood serum induced by peroral administration of propranolol might be due to the presence of this agent or its metabolites in the serum. It is noteworthy that in cell culture in vitro the atherogenicity of propranolol and other beta-blockers manifests itself at essentially high concentration of the drugs ( 10e5 M and higher). At the same time, peroral doses of propranolol close to therapeutic doses elicit atherogenic potential revealed in an ex vivo model [2]. The concentration-dependent differences of in vitro and ex vivo effects may be explained by the fact that ex vivo metabolites elicit an atherogeneous effect additionally to that of the mother molecule. It also cannot be excluded that ex vivo and in vitro effects may be governed by different mechanisms.
97*
9
?? 10
The results obtained in in vitro and ex vivo models suggest that in vivo the blood atherogenicity should manifest itself as cholesterol accumulation in arterial cells and stimulation of atherogenesis. This suggestion was checked in a model of atherosclerotic-like changes in the arterial wall caused by rabbit aorta injury induced by a balloon catheter. It was found that blood atherogenicity induced by long-term propranolol treatment is accompanied by lipid accumulation in the injury zone and by other atherosclerosisrelated changes, such as intimal thickening, hypercellularity and connective tissue growth. This is consistent with our in vitro and ex vivo findings demonstrating the stimulatory effect of betablockers on lipid accumulation and cellular proliferation [ 1,2,10,11]. On the other hand, we reported previously that cholesterol accumulation induced by atherogenic serum of patients is accompanied by all major manifestations of atherosclerosis at the cellular level: stimulation of proliferation, increased total protein synthesis and increased production of collagen, glycosaminoglycans and other extracellular matrix components [ 121. This suggests that propranololinduced intracellular accumulation of cholesterol can affect all other atherosclerotic indices. The effects of propranolol and other betablockers on the development of experimental atherosclerosis, including rabbit atherosclerosis, were examined by several research groups. Most of these investigations provide evidence in favor of
84
an antiatherogenic effect of propranolol which manifests itself as suppression of atherogenesis. However, there are studies demonstrating that beta-blockers are indifferent towards or even stimulate the atherosclerosis-related alterations in the vessel wall [for reviews see Refs. 13-151. The present study clearly demonstrates the propranolol atherogenicity which manifests itself as stimulation of all atherosclerotic indices in ballooned rabbit aorta. The discrepancy between our findings and. those reported by others can be explained by different experimental protocols. First, other researchers did not establish whether the betablocker examined generates an atherogenic potential in the blood of the animals. Second, in most studies experimental atherosclerosis was induced by hypercholesterolemic diets, while in our experiments it was induced by a mechanical injury to the aorta. Thus, we studied a cellular, lipid-poor lesion rather than a lipid-rich, foam cell lesion examined in other investigations. Finally, we employed a peroral administration of propranolol in contrast to intraperitoneal, subcutaneous, etc. used in other studies [ 16- 181. Loaldi et al. have recently reported that longterm peroral administration of propranolol aggravates coronary atherosclerosis in patients with angina of effort as compared with the calcium antagonists, nifedipine and isosorbide dinitrate [ 191. Nifedipine produced the best effect on coronary atherosclerosis by suppressing the development of existing and preventing the appearance of new atherosclerotic lesions. Isosorbide dinitrate was less effective in this respect, while with propranolol therapy the situation was the worst. These clinical observations agree with our in vitro results obtained in a cell model. Previously we demonstrated that calcium antagonists induce regression of atherosclerotic manifestations at the cellular level, beta-blockers stimulate these manifestations in a culture of aortic cells, and nitrates are without effect [2]. In addition, we have demonstrated that, in vitro, the atherogenicity of beta-blockers can be eliminated by combining them with nitrates or calcium antagonists. The atherogenic effect of propranolol revealed in the present study in vivo in an animal model is in agreement with the clinical observations of Loaldi et al. However, clinical trial was carried out
without a control group of patients who received placebo, therefore it is not clear whether propranolol is atherogenic at the background of naturally developing atherosclerosis. Presumably, beta-blockers do not aggravate but even impede the development of atherosclerosis in patients. Beta-blockers produce a broad spectrum of indirect beneficial effects which may slow down atherogenesis or induce its regression [14]. The resultant effect consisting of the negative atherogenic and positive antiatherogenic effects, in principle, can be positive. The fact that our ex vivo and in vitro data on the atherogenicity of beta-blockers were confirmed by clinical and in vivo observations motivated our attempt to eliminate this effect of propranolol. Our in vitro findings suggest that the atherogenicity of beta-blockers is eliminated by combination with calcium antagonists [2]. In some studies calcium antagonists suppress the development of experimental atherosclerosis and atherogenesis in patients as well as promote the regression of atherosclerotic lesions [ 19-241. We have demonstrated that in a culture of human aortic cells calcium antagonists produce antiatherosclerotic (regression of cellular manifestations) and antiatherogenic (prevention of lipid accumulation and other atherosclerotic manifestations) effects [ 10,111. There are powerful and weak calcium antagonists in this respect [2,11,24]. We have chosen papaverine which elicited a moderate antiatherosclerotic effect in cell culture [2]. This choice is explained by an attempt to employ a relatively weak agent as an indifferent additive to propranolol. Papaverine produced no effect on morphological and biochemical parameters reflecting the development of atherosclerotic-like alteration in rabbit aorta. On the other hand, papaverine prevented the appearance of serum atherogenicity induced by peroral administration of propranolol. Papaverine completely prevented the atherosclerotic changes induced by propranolol. We think that the results obtained are interesting from both theoretical and practical viewpoints. The atherogenic effects of beta-blockers revealed in vitro and ex vivo were confirmed in an in vivo model. In addition, the ability of calcium antagonists to eliminate the atherogenicity of beta-
85
blockers was demonstrated in vivo. From these findings one can conclude that the data obtained in our in vitro and ex vivo models correlate well with the in vivo situation. We hope that these models will be useful in the development of novel antiatherosclerotic drugs and optimization of direct antiatherosclerotic therapy. References Orekhov, A.N., Ruda, M.Ya., Baldenkov, G.N., Tertov, V.V., Khashimov, Kh.A., Li, H.R., Lyakishev, A.A., Kozlov, S.G., Tkachuk, V.A. and Smirnov, V.N.. Atherogenic effects of beta blockers on cells cultured from normal and atherosclerotic aorta, Am. J. Cardiol., 61 (1988) 1116. Orekhov, A.N., Baldenkov, G.N., Tertov, V.V.. Li, H.R.. Kozlov, S.G., Lyakishev, A.A., Tkachuk, V.A., Ruda, M.Ya. and Smirnov, V.N., Cardiovascular drugs and atherosclerosis: effects of calcium antagonists, betablockers and nitrates on atherosclerotic characteristics of human aortic cells, J. Cardiovasc. Pharmacol., I2 (1988) S66. Tertov, V.V., Orekhov. A.N., Nikitina, N.A., Perova. N.V., Lyakishev, A.A., Serebrennikov, S.G., Gratziansky, N.A., Nechaev, A.S. and Smirnov, V.N.. Peritoneal macrophages: a model for detecting atherogenic potential in patients’ blood serum, Ann. Med., 21 (1989) 455. Orekhov. A.N., Tertov, V.V.. Mukhin, D.N., Koteliansky, V.E., Glukhova, M.A., Frid, M.G., Sukhova, G.K., Khashimov. Kh.A. and Smirnov, V.N.. Insolubilization of low density lipoprotein induces cholesterol accumulation in cultured subendothelial cells of human aorta, Atherosclerosis, 79 (1989) 59. Orekhov, A.N., Tertov. V.V.. Kudryashov. S.A.. Khashimov, Kh.A. and Smirnov, V.N., Primary culture of human aortic intima cells as a model for testing antiatherosclerotic drugs. Effects of cyclic AMP, prostaglandins, calcium antagonists, antioxidants and lipid-lowering agents, Atherosclerosis, 60 (1986) 101. Clowes, A.W., Reidy, M.A. and Clowes. M.M., Mechanisms of stenosis after arterial injury, Lab. Invest., 49 (1983) 208. Orekhov, A.N., Tertov, V.V., Novikov, I.D., Krushinsky, A.V., Andreeva, E.R., Lankin, V.Z. and Smimov, V.N., Lipids in cells of atherosclerotic and uninvolved human aorta. I. Lipid composition of aortic tissue and enzyme isolated and cultured cells, Exp. Mol. Pathol., 42 (1985) 117. Orekhov, A.N., Andreeva, E.R., Krushinsky, A.V.. Novikov, I.D., Tertov, V.V., Nestaiko, G.V., Khashimov, Kh.A., Repin, V.S. and Smirnov, V.N., Intimal cells and atherosclerosis: relationship between the number of intimal cells and major manifestations of atherosclerosis in human aorta, Am. J. Pathol., 125 (1986) 402. Dixon, W.J. and Brown, M.B., Biomedical Computer Programs. P-Series. Berkeley, University of California Press, 1977, p. 185.
IO Orekhov, A.N., Baldenkov, G.N., Tertov, V.V., Ruda, M.Ya., Khashimov, Kh.A., Kudryashov, S.A., Li, H.R., Kozlov, S.G., Lyakishev, A.A., Tkachuk, V.A. and Smirnov, V.N., Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture, Herz, 15 (1990) 139. II Orekhov, A.N., In vitro models of antiatherosclerotic effects of cardiovascular drugs, Am. J. Cardiol., 66 (1990) 231. 12 Orekhov, A.N., Tertov, V.V., Kudryashov, S.A. and Smirnov, V.N., Triggerlike stimulation of cholesterol accumulation and DNA and extracellular matrix synthesis induced by atherogenic serum or low density lipoprotein in cultured cells, Circ. Res., 66 (1990) 311. 13 Kaplan, J.R., Manuck, S.B., Adams, M.R. and Clarkson, T.B., The effects of beta-adrenergic blocking agents on atherosclerosis and its complications, Eur. Heart. J., 8 (1987) 928. 14 Aablad, B., Bjorkman, J.-A., Gustafsson, D., Hansson, G., Ostlund-Lindqvist, A.-M. and Pettersson, K. The role of sympathetic activity in atherogenesis effects of Bblockade, Am. Heart J., 116 (1988) 322. 15 Cruickshank, J.M. and Smith, J.C., The beta-receptor, atheroma and cardiovascular damage, Pharmacol. Ther., 42 (1989) 385. 16 Whittington-Coleman, P.J., Carrier, O., Jr. and Douglas, B.H. The effects of propranolol on cholesterol-induced atheromatous lesions, Atherosclerosis. I8 (1973) 337. 17 Chobanian, A.V., Brecher, P. and Chart, C., Effects of propranolol on atherogenesis in the cholesterol-fed rabbit, Circ. Res., 56 (1985) 755. 18 Ostlund-Lindqvist, A.-M., Lindqvist, P., Brautigam, J., Olsson, G., Bondjers, G. and Nordborg, C., Effect of metoprolol on diet-induced atherosclerosis in rabbits, Arteriosclerosis, 8 (1988) 40. 19 Loaldi, A., Polese, A., Montorsi, P., De Cesare, N., Fabbiocchi, F., Ravagnani, P. and Guazzi, M.D., Comparison of nifedipine, propranolol and isosorbide dinitrate on angiographic progression and regression of coronary arterial narrowings in angina pectoris. Am. J. Cardiol., 64 (1989) 433. 20 Henry, P.D., Atherogenesis, calcium and calcium antagonists, Am. J. Cardiol., 66 (1990) 31. 21 Parmley, W.W., Vascular protection from atherosclerosis: potential of calcium antagonists, Am. J. Cardiol., 66 (1990) 161. 22 Kober, G., Schneider, W. and Kaltenbach, M. Can the progression of coronary sclerosis be influenced by calcium antagonists? J. Cardiovasc. Pharmacol., 13 (1989) S2. 23 Lichtlen, P.R., Hugenholtz, P.G., Rafflenbeul, W., Hecker, H.. Jost, S. and Decker% J.W. on behalf of the Retardation of INTACT investigators, group angiographic progression of coronary artery disease by nifedipine. Results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT), Lancet, 335 (1990) 1109. 24 Baldenkov, G.N.. Akopov, S.E., Li, H.R. and Orekhov, A.N., Prostacyclin, thromboxane A, and calcium antagonists: effects on atherosclerotic characteristics of vascular cells, Biomed. Biochim. Acta, 47 (1988) S324.
Printed and Published in Ireland
ATHERO 04854
Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine Alexander N. Orekhov, Irina V. Andrianova, Marc D. Rekhter, Vladimir V. Tertov, Elena R. Andreeva, Sergey E. Ragimov and Alexander A. Mironov Institute of Experimental Cardiology, National Cardiology Research Center, 3rd Cherepkovskaya Street I5 A, 121552 Moscow (Russia)
(Received 27 January, 1992) (Revised, received 7 April, 1992) (Accepted 8 April, 1992)
Summary The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally refered to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate betablocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy. Key words: Atherosclerosis; Beta-blockers; Calcium antagonists; Cell culture; Foam cells; Macrophages; Rabbit aorta injury; Smooth muscle cells
Correspondence IO: Alexander N. Orekhov, Ph.D., Institute of Experimental Cardiology, National Cardiology Research Center, 3rd Cherepkovskaya Street 15 A, 121552 Moscow, Russia. Tel.: (095) 149-0559; Fax: (095) 415-2962, (095) 4146699.
Introduction We have recently reported that the betablockers propranolol, alprenolol, metoprolol,
78
atenolol, pindolol and timolol increase intracellular cholesterol content and stimulate proliferative activity of smooth muscle cells cultured from human aortic intima [ 1,2]. These data allowed us to suggest that beta-blockers elicit a direct atherogenic effect at the arterial cell level. Since intracellular lipid administration and stimulation of cell proliferation are the earliest manifestations of atherosclerosis at the cell level, the ability of betablockers to stimulate these processes in vitro was conventionally termed an atherogenic potential or [ 1,2]. Atherogenicity of proatherogenicity pranolol was also revealed when blood sera of patients were examined. Blood serum obtained after a peroral administration of the drug stimulated proliferation of cultured cells and induced accumulation of intracellular cholesterol [ 1,2]. The atherogenicity of beta-blockers elicited in a cell culture can be considerably reduced or eliminated by combining them with other drugs. For example, nitroglycerin and the calcium antagonist, nifedipine, abolished the stimulation of cell proliferation and intracellular accumulation of cholesterol induced by metoprolol [2]. The data mentioned were obtained on models in vitro (addition of the drug tested to cell culture) or ex vivo (addition of patient’s blood serum after drug administration to cell culture). A question arises whether the atherogenicity of beta-blockers occurs in vivo. If so, can this atherogenicity be eliminated by simultaneous administration of beta-blockers with other drugs? The answer to this question can be provided by in vivo investigations. In this study rabbits were used to find out whether the atherogenicity of beta-blockers is elicited after a peroral administration of these drugs. After the atherogenicity of beta-blockers had been revealed in vitro, ex vivo and in vivo, we attempted to eliminate the atherogenicity of the beta-blocker, propranolol, by combining it with papaverine. Materials and Methods
Propranolol (Anaprilin) and papaverine were purchased from Zdorov’e ChemicoPharmaceutical Corporation (Kharkov, USSR). Metoprolol (Spesicor) was from Laaketehdas Leiras Huhtamaki Oy (Turku, Finland), atenolol (Tenolol) from IPCA Laboratories Ltd. (Bombay,
India), pindolol (Visken) from EGIS (Budapest, Hungary) and timolol (Ocupres-E) from Cadila Laboratories Pvt. Ltd. (Ahmedabad, India). For in vitro experiments, alprenolol hydrochloride and metoprolol bitartrate were from Astra Pharmaceuticals Production AB (Sodertalje, Sweden); papaverine hydrochloride, propranolol hydrochloride, pindolol, atenolol, timolol maleate were purchased from Sigma Chemical Company (St. Louis, MO). Experiments were performed on male 12-15week Chinchilla rabbits weighing 3.0-3.5 kg. Animals were maintained on standard diet. Rabbit blood serum was added to cultured peritoneal macrophages of BALB/c mice obtained as described [3]. Cells were washed with Medium 199 and incubated for 4 h in medium 199 containing glutamine, antibiotics and 10% of the serum tested. Cells were extensively washed and the total cholesterol content was determined as previously 141. Human aortic subendothelial intimal smooth muscle cells were isolated from autopsy obtained within 2-3 h after death. Cells were harvested by enzymatic digestion of grossly normal part of aortic intima with 0.1% collagenase for 3-4 h and seeded into 24-well culture plates. Intimal cells were cultured for 7 days in Medium 199, containing 10% fetal calf serum, glutamine and antibiotics. On the seventh day of cultivation, test agents were added and total cholesterol content was determined in 24 h. Protocols for cell isolation and cultivation as well as technical details of the experiment are presented elsewhere [5]. Injury of rabbit aorta was produced under Nembutal anesthesia. The abdominal aorta was deendothelized with a 4F balloon catheter (61. After the operation, rabbits were assigned into 4 groups of 7-8 animals each. Group I animals received propranolol perorally at a dose of 20 mg, group II 20 mg papaverine, group III propranolol and papaverine and group IV 1 ml of distilled water. The drugs were dissolved in 1 ml of distilled water and given 3 times a day (at lO:OO,14:00 and 18:00 h) during a 21-day period. The whole experiment was repeated 3 times independently. The aorta was dissected on day 21 after deendothelization. A strip of aorta (5 mm wide) from the central part of the injury was fixed with 2.5%
79
glutaraldehyde for morphological examination. The thickness of media and intima was measured in semi-thin sections under a light microscope with the aid of a micrometer. The remainder of the aorta was used in biochemical studies. Neointima and media were separated mechanically, lipids were extracted with a chloroformmethanol mixture (2: 1) and separated by thin-layer chromatography. Phospholipids, free cholesterol, cholesterol esters and triglycerides ‘were quantitated by scanning densitometry [7]. The collagen content and cell number in the neointima were determined as described elsewhere [8]. The significance of differences was evaluated by dispersion analysis methods using a BMDP statistical program package 19). Results
Atherogenicity of beta-blockers In vitro. After 24 h of incubation with primary culture of human aortic intimal cells, the betablockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol induced a considerable increase in the total intracellular cholesterol content (Table 1). This confirms our previous observations [ 1,2]. Similar results were obtained in a culture of mouse peritoneal macrophages: after 4 h of incubation with beta-blockers intracellular cholesterol content increased by 30-90% (Table 1). Since the atherogenic effects of beta-blockers originally revealed in cultured human aortic cells are readily reproduced in cultured mouse peritoneal macrophages and taking into account that experiments on mouse macrophages are less laborious than those on human cells, in subsequent studies we employed a culture of mouse peritoneal macrophages. Ex vivo. Serum prepared from rabbit blood obtained 1,2,3,4 and 5 h after peroral administration of beta-blockers was added to a culture of peritoneal macrophages. Serum obtained 1 h after propranolol induced a substantial accumulation of cholesterol in the cells, i.e. exhibited atherogenicity (Fig. 1). Atherogenicity of the serum obtained 2 h after the drug administration was maximal and was retained during 4 h (Fig. 1). Similar effects were observed with blood sera obtained after peroral administration of metopro-
TABLE 1 CHOLESTEROL CONTENT WITH BETA-BLOCKERS
IN
CELLS
CULTURED
All drugs were tested in concentration of lo-’ M. Data are expressed as mean of 4 determinations f S.E.M. Cells were cultured in Medium 199, containing 10% fetal calf serum, glutamine and antibiotics as described in Materials and Methods. Control levels of total cholesterol in cultured cells were 53 * 4 pglmg cell protein (aortic cells) and 26 ?? 2 &mg cell protein (macrophages). n.a., not assayed. Drug
None (control) Propranolol Metoprolol Atenolol Pindolol Alprenolol Timolol
Cholesterol content (% of control) Human aortic intimal smooth muscle cells
Mouse peritoneal macrophages
loo* 158 zt 144 * 139 f 140 f 151 * 154 f
loo* 8 187 zt 11’ 182 ?? 10” 187 zt lO* n.a. 154 f 18* 132 f 9*
3 5’ 7* 9* 8’ 108 8*
*Significant difference from control, P < 0.05.
propranolol
Fig. 1. Effect of peroral administration of propranolol and propranolol-papaverine combination on the ability of rabbit serum to induce cholesterol accumulation in cultured macrophages. Propranolol (20 mg) or combination of propranolol (20 mg) plus papaverine (20 mg) were administered perorally. The time of administration is indicated with an arrow. Blood was collected 1,2, 3,4 and 5 h after administration. Blood serum obtained before (0 h) and after the drug administration were added to a culture of mouse peritoneal macrophages at a final concentration of 10%. The intracellular cholesterol content was determined 4 h afterwards. The cholesterol content in cells incubated with blood serum obtained before the drug administration was assumed as 100%. Each point represents the mean of 6-8 determinations in separate experiments.
80 TABLE 2 CHOLESTEROL CONTENT IN MOUSE PERITONEAL MACROPHAGES CULTURED WITH BLOOD SERUM OF RABBITS TREATED WITH BETA-BLOCKERS Data are expressed as mean of 4 determinations f S.E.M. Blood was drawn 2 h after oral drug administration and blood serum was added to cultured mouse peritoneal macrophages. Cell cholesterol content was measured 4 h later. Drug
Dose (mg)
Cholesterol content (“h of control)
None (control) Propranolol Metoprolol Atenolol Pindolol Timolol
-
100 195 f 126 * 161 zt 129 zt 194 f
20 50 100 5 2.5
5* 9* IO* 8* 19*
*Significant difference from control, P < 0.05.
101, atenolol, pindolol and timolol. These agents induced serum atherogenicity which manifested itself as cholesterol accumulation in cultured macrophages (Table 2). The beta-blocker propranolol was chosen for a detailed examination since it induced maximum atherogenicity both in vitro and ex vivo. Two hours after the first dose of propranolol(20 mg) rabbit blood serum became atherogenic,
i.e. it induced a 2.5fold accumulation of intracellular cholesterol (Fig. 2). Four hours after the first dose, blood atherogenicity was slightly decreased, remaining, however, at an essentially high level and it increased after the second dose (Fig. 2). By 4 h after the second dose (8 h after the first dose) serum atherogenicity decreased again but it was elevated after the third dose (Fig. 2). Twenty-four hours after the first dose followed by two doses given at 4-h intervals, serum atherogenicity remained high and increased a little after each subsequent dose (Fig. 2). Regular peroral administration of propranolol at 4-h intervals for 21 days maintained the atherogenicity of rabbit serum at a high level during the entire period (Fig. 3). In viva In intact rabbit aorta the intima was not pronounced (Fig. 4). Injury produced with a balloon catheter induced a considerable thickening of intima, i.e. formation of neointima (Fig. 4). Microscopically, 21 days after ballonization an elevation was detected in the injury zone in all aortas. On average, the thickness of neointima was one half of that of the media. The thickness of neointima in the aorta of control rabbits and rabbits given propranolol was compared in three independent experiments. Visually, the neointima of propranolol-treated rabbits was considerably thicker than that of control animals (Fig. 4). On
6
a
fg w_, 2 E 150-
Pz
Oal@ 3
0”
----------------------
=O- I 0
I
I 8
I
18
I 24
c
3;
HOURS Fig. 2. Effect of three peroral doses of propranolol on atherogenicity of rabbit blood serum. The atherogenicity of blood serum displayed as stimulation of cholesterol accumulation in cultured macrophages was evaluated as described in the legend to Fig. 1. The time of propranolol administration are indicated with arrows. Other details are the same as in the legends to Fig. 1.
0
5ot
I
0
I
10
I
20
DAYS Fig. 3. Effect of a long-term propranolol treatment on the atherogenicity of rabbit blood serum. Blood was collected on day 1, 2, 3, 7, 14 and 21 in the morning before and 2 h after the drug administration. Other details are the same as in the legends to Fig. 1.
82 TABLE 3 INFLUENCE OF PAPAVERINE ON PROPRANOLOL Parameter
Intima:media ratio Cell number cells/mg wet weight Cholesteryl esters &mg dry weight Free cholesterol pg/mg dry weight Triglycerides &mg dry weight Phospholipids pglmg dry weight Collagen &mg wet weight
Control values (intact rabbit aortas)
ATHEROGENICITY
% of control Control
Propranolol
Papaverine
0.48 + 0.01
loo
203 f 20*
96 f 8
94 f 8
(14.2 zt 1.2) x IO3
100
305 f 99
II6 f 4
127 zt 8
loo
573
107 ?? I1
94zt 16
27.7 zk 2.3
100
244 zt 32*
124 f I3
95 f IO
11.1 f 1.3
loo
355
?? 31*
161 + 38
158 f 26
21.9 zt 0.9
100
198 f 20.
n.a.
n.a.
331 f 26
100
172 f 20*
104 + 8
1.7
?? 0.4
?? 1358
Propranolol + papaverine
99*
IO
*Significant difference from control, P < 0.05. The data are calculated per intimal surface.
eliminated the atherogenicity of propranolol in vitro. Ex viva Simultaneous peroral administration of propranolol (20 mg) and papaverine (20 mg and more) prevented the appearance of atherogenic properties of rabbit blood serum (Fig. 1). Papaverine at a dose of 10 mg and lower did not eliminate the atherogenicity of propranolol completely but retarded its manifestations (Table 5). Regular administration of the propranololTABLE 4 CHOLESTEROL CONTENT IN MOUSE PERITONEAL MACROPHAGES CULTURED WITH PROPRANOLOL AND PAPAVERINE Details are the same as in Table 1 Drug
Cholesterol accumulation (% of control)
None (control) Propranolol Papaverine Propranolol + papaverine
100 zt 187 zt 102 f 93 zt
8 Ii+ I2 8
?? Significant difference from control, P < 0.05,
papaverine combination during 21 days resulted in a complete elimination of the propranolol atherogenicity (Fig. 3). Thus, papaverine abolished the atherogenic effect of propranolol applied in a single dose and in a long-term treatment. It should be mentioned that propranolol, papaverin, or propranolol-papaverin combination did not produce any effect on the blood cholesterol content during a 21-day period (not illustrated). In viva. Aortic neointima of rabbits given propranolol alone was considerably thicker than that of rabbits given propranolol with papaverine. Visually, the latter was similar to the neointima of control animals (Fig. 4). Accurate measurements confirmed the visual observations: on average the thickness of the neointima of rabbits given propranolol-papaverine was 2-fold less than that of propranolol-treated rabbits and equal to that of controls (Table 3). Papaverine alone had no effect on the intima thickness (Table 3). Papaverine completely prevented the propranolol-induced increase in the cell number and in lipid and collagen contents (Table 3). Papaverine by itself produced no effect on these indices (Table 3).
83 TABLE 5 EFFECT OF PROPRANOLOL AND PROPRANOLOL-PAPAVERINE CULTURED MACROPHAGES
COMBINATION ON CHOLESTEROL CONTENT IN
Blood serum obtained before (0 h) and after peroral drug administration was added to a culture of mouse peritoneal macrophages at a final concentration of 10%. The intracellular cholesterol content was determined 4 h afterwards. The cholesterol content in cells incubated with blood serum obtained before the drug administration was assumed as 100%. Other details are the same as in Table 2. Hours after administration
Administration
Propranolol, 20 Propranolol, 20 + papaverine, Propranolol, 20 + papaverine,
mg mg 10 mg mg 20 mg
0
1
2
3
4
100 ?? 10 loo* 5
145 zt 18 121 f 10
190 f 19* 134 f 14
176 zt 18; 174 f 14*
135 ?? 15’ 140 + 5*
loo*
105 ?? 17
loo
100 ?? 11
5
Discussion of mouse peritoneal Using a culture macrophages, we have confirmed our previous findings that beta-blockers are atherogenic at the cellular level [ 1,2], All beta-blockers examined (propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol) induced cholesterol accumulation in cultured cells. Peroral administration of these beta-blockers generated an atherogenie potential in rabbit blood serum, i.e., they acquired the ability to induce intracellular cholesterol accumulation. Long-term propranolol treatment produced a sustained blood atherogenicity The atherogenic potential of blood serum induced by peroral administration of propranolol might be due to the presence of this agent or its metabolites in the serum. It is noteworthy that in cell culture in vitro the atherogenicity of propranolol and other beta-blockers manifests itself at essentially high concentration of the drugs ( 10e5 M and higher). At the same time, peroral doses of propranolol close to therapeutic doses elicit atherogenic potential revealed in an ex vivo model [2]. The concentration-dependent differences of in vitro and ex vivo effects may be explained by the fact that ex vivo metabolites elicit an atherogeneous effect additionally to that of the mother molecule. It also cannot be excluded that ex vivo and in vitro effects may be governed by different mechanisms.
97*
9
?? 10
The results obtained in in vitro and ex vivo models suggest that in vivo the blood atherogenicity should manifest itself as cholesterol accumulation in arterial cells and stimulation of atherogenesis. This suggestion was checked in a model of atherosclerotic-like changes in the arterial wall caused by rabbit aorta injury induced by a balloon catheter. It was found that blood atherogenicity induced by long-term propranolol treatment is accompanied by lipid accumulation in the injury zone and by other atherosclerosisrelated changes, such as intimal thickening, hypercellularity and connective tissue growth. This is consistent with our in vitro and ex vivo findings demonstrating the stimulatory effect of betablockers on lipid accumulation and cellular proliferation [ 1,2,10,11]. On the other hand, we reported previously that cholesterol accumulation induced by atherogenic serum of patients is accompanied by all major manifestations of atherosclerosis at the cellular level: stimulation of proliferation, increased total protein synthesis and increased production of collagen, glycosaminoglycans and other extracellular matrix components [ 121. This suggests that propranololinduced intracellular accumulation of cholesterol can affect all other atherosclerotic indices. The effects of propranolol and other betablockers on the development of experimental atherosclerosis, including rabbit atherosclerosis, were examined by several research groups. Most of these investigations provide evidence in favor of
84
an antiatherogenic effect of propranolol which manifests itself as suppression of atherogenesis. However, there are studies demonstrating that beta-blockers are indifferent towards or even stimulate the atherosclerosis-related alterations in the vessel wall [for reviews see Refs. 13-151. The present study clearly demonstrates the propranolol atherogenicity which manifests itself as stimulation of all atherosclerotic indices in ballooned rabbit aorta. The discrepancy between our findings and. those reported by others can be explained by different experimental protocols. First, other researchers did not establish whether the betablocker examined generates an atherogenic potential in the blood of the animals. Second, in most studies experimental atherosclerosis was induced by hypercholesterolemic diets, while in our experiments it was induced by a mechanical injury to the aorta. Thus, we studied a cellular, lipid-poor lesion rather than a lipid-rich, foam cell lesion examined in other investigations. Finally, we employed a peroral administration of propranolol in contrast to intraperitoneal, subcutaneous, etc. used in other studies [ 16- 181. Loaldi et al. have recently reported that longterm peroral administration of propranolol aggravates coronary atherosclerosis in patients with angina of effort as compared with the calcium antagonists, nifedipine and isosorbide dinitrate [ 191. Nifedipine produced the best effect on coronary atherosclerosis by suppressing the development of existing and preventing the appearance of new atherosclerotic lesions. Isosorbide dinitrate was less effective in this respect, while with propranolol therapy the situation was the worst. These clinical observations agree with our in vitro results obtained in a cell model. Previously we demonstrated that calcium antagonists induce regression of atherosclerotic manifestations at the cellular level, beta-blockers stimulate these manifestations in a culture of aortic cells, and nitrates are without effect [2]. In addition, we have demonstrated that, in vitro, the atherogenicity of beta-blockers can be eliminated by combining them with nitrates or calcium antagonists. The atherogenic effect of propranolol revealed in the present study in vivo in an animal model is in agreement with the clinical observations of Loaldi et al. However, clinical trial was carried out
without a control group of patients who received placebo, therefore it is not clear whether propranolol is atherogenic at the background of naturally developing atherosclerosis. Presumably, beta-blockers do not aggravate but even impede the development of atherosclerosis in patients. Beta-blockers produce a broad spectrum of indirect beneficial effects which may slow down atherogenesis or induce its regression [14]. The resultant effect consisting of the negative atherogenic and positive antiatherogenic effects, in principle, can be positive. The fact that our ex vivo and in vitro data on the atherogenicity of beta-blockers were confirmed by clinical and in vivo observations motivated our attempt to eliminate this effect of propranolol. Our in vitro findings suggest that the atherogenicity of beta-blockers is eliminated by combination with calcium antagonists [2]. In some studies calcium antagonists suppress the development of experimental atherosclerosis and atherogenesis in patients as well as promote the regression of atherosclerotic lesions [ 19-241. We have demonstrated that in a culture of human aortic cells calcium antagonists produce antiatherosclerotic (regression of cellular manifestations) and antiatherogenic (prevention of lipid accumulation and other atherosclerotic manifestations) effects [ 10,111. There are powerful and weak calcium antagonists in this respect [2,11,24]. We have chosen papaverine which elicited a moderate antiatherosclerotic effect in cell culture [2]. This choice is explained by an attempt to employ a relatively weak agent as an indifferent additive to propranolol. Papaverine produced no effect on morphological and biochemical parameters reflecting the development of atherosclerotic-like alteration in rabbit aorta. On the other hand, papaverine prevented the appearance of serum atherogenicity induced by peroral administration of propranolol. Papaverine completely prevented the atherosclerotic changes induced by propranolol. We think that the results obtained are interesting from both theoretical and practical viewpoints. The atherogenic effects of beta-blockers revealed in vitro and ex vivo were confirmed in an in vivo model. In addition, the ability of calcium antagonists to eliminate the atherogenicity of beta-
85
blockers was demonstrated in vivo. From these findings one can conclude that the data obtained in our in vitro and ex vivo models correlate well with the in vivo situation. We hope that these models will be useful in the development of novel antiatherosclerotic drugs and optimization of direct antiatherosclerotic therapy. References Orekhov, A.N., Ruda, M.Ya., Baldenkov, G.N., Tertov, V.V., Khashimov, Kh.A., Li, H.R., Lyakishev, A.A., Kozlov, S.G., Tkachuk, V.A. and Smirnov, V.N.. Atherogenic effects of beta blockers on cells cultured from normal and atherosclerotic aorta, Am. J. Cardiol., 61 (1988) 1116. Orekhov, A.N., Baldenkov, G.N., Tertov, V.V.. Li, H.R.. Kozlov, S.G., Lyakishev, A.A., Tkachuk, V.A., Ruda, M.Ya. and Smirnov, V.N., Cardiovascular drugs and atherosclerosis: effects of calcium antagonists, betablockers and nitrates on atherosclerotic characteristics of human aortic cells, J. Cardiovasc. Pharmacol., I2 (1988) S66. Tertov, V.V., Orekhov. A.N., Nikitina, N.A., Perova. N.V., Lyakishev, A.A., Serebrennikov, S.G., Gratziansky, N.A., Nechaev, A.S. and Smirnov, V.N.. Peritoneal macrophages: a model for detecting atherogenic potential in patients’ blood serum, Ann. Med., 21 (1989) 455. Orekhov. A.N., Tertov, V.V.. Mukhin, D.N., Koteliansky, V.E., Glukhova, M.A., Frid, M.G., Sukhova, G.K., Khashimov. Kh.A. and Smirnov, V.N.. Insolubilization of low density lipoprotein induces cholesterol accumulation in cultured subendothelial cells of human aorta, Atherosclerosis, 79 (1989) 59. Orekhov, A.N., Tertov. V.V.. Kudryashov. S.A.. Khashimov, Kh.A. and Smirnov, V.N., Primary culture of human aortic intima cells as a model for testing antiatherosclerotic drugs. Effects of cyclic AMP, prostaglandins, calcium antagonists, antioxidants and lipid-lowering agents, Atherosclerosis, 60 (1986) 101. Clowes, A.W., Reidy, M.A. and Clowes. M.M., Mechanisms of stenosis after arterial injury, Lab. Invest., 49 (1983) 208. Orekhov, A.N., Tertov, V.V., Novikov, I.D., Krushinsky, A.V., Andreeva, E.R., Lankin, V.Z. and Smimov, V.N., Lipids in cells of atherosclerotic and uninvolved human aorta. I. Lipid composition of aortic tissue and enzyme isolated and cultured cells, Exp. Mol. Pathol., 42 (1985) 117. Orekhov, A.N., Andreeva, E.R., Krushinsky, A.V.. Novikov, I.D., Tertov, V.V., Nestaiko, G.V., Khashimov, Kh.A., Repin, V.S. and Smirnov, V.N., Intimal cells and atherosclerosis: relationship between the number of intimal cells and major manifestations of atherosclerosis in human aorta, Am. J. Pathol., 125 (1986) 402. Dixon, W.J. and Brown, M.B., Biomedical Computer Programs. P-Series. Berkeley, University of California Press, 1977, p. 185.
IO Orekhov, A.N., Baldenkov, G.N., Tertov, V.V., Ruda, M.Ya., Khashimov, Kh.A., Kudryashov, S.A., Li, H.R., Kozlov, S.G., Lyakishev, A.A., Tkachuk, V.A. and Smirnov, V.N., Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture, Herz, 15 (1990) 139. II Orekhov, A.N., In vitro models of antiatherosclerotic effects of cardiovascular drugs, Am. J. Cardiol., 66 (1990) 231. 12 Orekhov, A.N., Tertov, V.V., Kudryashov, S.A. and Smirnov, V.N., Triggerlike stimulation of cholesterol accumulation and DNA and extracellular matrix synthesis induced by atherogenic serum or low density lipoprotein in cultured cells, Circ. Res., 66 (1990) 311. 13 Kaplan, J.R., Manuck, S.B., Adams, M.R. and Clarkson, T.B., The effects of beta-adrenergic blocking agents on atherosclerosis and its complications, Eur. Heart. J., 8 (1987) 928. 14 Aablad, B., Bjorkman, J.-A., Gustafsson, D., Hansson, G., Ostlund-Lindqvist, A.-M. and Pettersson, K. The role of sympathetic activity in atherogenesis effects of Bblockade, Am. Heart J., 116 (1988) 322. 15 Cruickshank, J.M. and Smith, J.C., The beta-receptor, atheroma and cardiovascular damage, Pharmacol. Ther., 42 (1989) 385. 16 Whittington-Coleman, P.J., Carrier, O., Jr. and Douglas, B.H. The effects of propranolol on cholesterol-induced atheromatous lesions, Atherosclerosis. I8 (1973) 337. 17 Chobanian, A.V., Brecher, P. and Chart, C., Effects of propranolol on atherogenesis in the cholesterol-fed rabbit, Circ. Res., 56 (1985) 755. 18 Ostlund-Lindqvist, A.-M., Lindqvist, P., Brautigam, J., Olsson, G., Bondjers, G. and Nordborg, C., Effect of metoprolol on diet-induced atherosclerosis in rabbits, Arteriosclerosis, 8 (1988) 40. 19 Loaldi, A., Polese, A., Montorsi, P., De Cesare, N., Fabbiocchi, F., Ravagnani, P. and Guazzi, M.D., Comparison of nifedipine, propranolol and isosorbide dinitrate on angiographic progression and regression of coronary arterial narrowings in angina pectoris. Am. J. Cardiol., 64 (1989) 433. 20 Henry, P.D., Atherogenesis, calcium and calcium antagonists, Am. J. Cardiol., 66 (1990) 31. 21 Parmley, W.W., Vascular protection from atherosclerosis: potential of calcium antagonists, Am. J. Cardiol., 66 (1990) 161. 22 Kober, G., Schneider, W. and Kaltenbach, M. Can the progression of coronary sclerosis be influenced by calcium antagonists? J. Cardiovasc. Pharmacol., 13 (1989) S2. 23 Lichtlen, P.R., Hugenholtz, P.G., Rafflenbeul, W., Hecker, H.. Jost, S. and Decker% J.W. on behalf of the Retardation of INTACT investigators, group angiographic progression of coronary artery disease by nifedipine. Results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT), Lancet, 335 (1990) 1109. 24 Baldenkov, G.N.. Akopov, S.E., Li, H.R. and Orekhov, A.N., Prostacyclin, thromboxane A, and calcium antagonists: effects on atherosclerotic characteristics of vascular cells, Biomed. Biochim. Acta, 47 (1988) S324.
