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The effects of propranolol on cholesterol-induced atheromatous lesions
Atherosclerosis, 18 (1973) 337-345 0 Elsevier Scientific Publishing Company,
THE
EFFECTS
ROMATOUS
337 Amsterdam
OF PROPRANOLOL
- Printed in The Netherlands
ON CHOLESTEROL-INDUCED
ATHE-
LESIONS
PEGGY J. WHITTINGTON-COLEMAN*, GLAS*** Departments of Medicine and Pharmacology, 39216 (U.S.A.)
(Received November
OLIVER
CARRIER,
JR.**
AND
BEN H. DOU-
University of Mississippi Medical Center, Jackson, Miss.
14th, 1972)
SUMMARY
The effects of propranolol on cholesterol-induced atheromatous lesions in rabbits and on the calcium content of the vascular tissue of these animals were studied. The calcium content of normal rabbit aortae was significantly lower than that of the aortae of rabbits receiving a 2 % cholesterol supplemented diet. The calcium content from rabbit aortae which had received a 2% by weight cholesterol diet was significantly higher than that of the aortae of rabbits which received propranolol plus cholesterol, and of the aortae of rabbits which received a normal diet plus propranolol. Histological results showed atheromatous plaques within the intimal layer of the aortae of the cholesterol group. Some of the rabbits which received propranolol plus cholesterol had lesions, but these were less severe than those of the cholesterol group.
These results indicate
that propranolol
affects fat and calcium
metabolism
in
the aorta.
Key words :
Supported
Atheromatous
lesions
-
Propranolol - Serum
cholesterol
in patt by U.S. Public Health Service Grants HE-09031 and HE-09192. Postdoctoral Fellow and formerly NIH Predoctoral trainee (GM-39063). ** Present address: University of Texas at San Antonio, San Antonio, Texas, U.S.A. *** Established Investigator, American Heart Association. Please address all correspondence and requests for reprints to: Dr. Peggy J. WhittingtonColeman, Department of Medicine, University of Mississippi Medical Center, Jackson, Miss. 39216, U.S.A.
* Mississippi Heart Association
338
P. J. WHITTINGTON-COLEMAN,
0.
CARRIER,
JR.,
B. H. DOUGLAS
INTRODUCTION
Investigations in this laboratory have shown that cholesterol induced atherosclerosis in rabbits results in an increase in the total calcium and lipids of the sclerotic aortaeiy2. We have also shown that hypotensive agents such as reserpine, guanethidine and the reserpine
analogue,
methyl-0-reserpate
(SU-7064),
as well as the reserpine
analogue with no hypotensive properties (methyl-18-epireserpate methyl ether), prevent a rise in aortic calcium and the development of atherosclerotic lesions in rabbits fed a high cholesterol dietiJ. It has been demonstrated that propranolol depresses the sarco-tubular calcium pump4 in concentrations that depress cardiac contractilitys. Bohr6 has suggested that the beta-blockers decrease the amount of ionized calcium available to the cell. Prichard and Gillam7,s reported that propranolol produced a slight but significant degree of hypotension
in humans.
Since propranolol
apparently
affects both
calcium
meta-
bolism and has hypotensive properties it appeared conceivable that it could have a protective action against diet-induced atherosclerosis either by decreasing the blood pressure or by some mechanism involving calcium metabolism. The subject of the present report is our observations on the effect of propranolol on the development of the atherosclerotic lesion. METHODS
For this study 36 albino rabbits of either sex, weighing between 2.0 and 2.5 kg were used. Ten of the rabbits were maintained on a normal Purina diet throughout the experiment. Group 1 (10 animals)
received a normal
Purina
rabbit
pellet diet supplemented
with 2% cholesterol by weight. Group 2 (8 animals) received the same dietary regimen as Group 1, but were additionally treated with 5 mg of propranolol/kg/day (Inderal) administered intraperitoneally from the beginning of the experiment. Group 3 (8 animals) received a normal Purina rabbit pellet diet and were additionally treated with 5 mg of propranolol/kg/day administered intraperitoneally. Systolic blood pressures were recorded three times a week from an ear artery by a Grant-Rothschild capsule. The animals had ad libitum access to diet and water. Food consumption was followed throughout the experiment. Total body weight measurements were made weekly until the experiment was terminated. Blood samples were drawn from an ear artery at the beginning of the experiment, after 5 weeks and when the experiment terminated. Serum cholesterol levels were determined by a modified Lieberman-Buchard reaction. The animals were sacrificed by a sharp blow on the head 10 weeks after onset of the experimental feeding period. The aortae were removed from the aortic valve to the bifurcation. After the aortae had been removed from the animals they were cleaned of blood and divided into three sections. Section one consisted of the aortic
339
PROPRANOLOL IN ATHEROMATOUS LESIONS
arch from the aortic valve to the first pair of intercostal intercostal
arteries
to the coeliac artery
and section
arteries,
section two from the
three from the coeliac artery to
the bifurcation. The proximal portion of each section (approximately removed and analyzed for calcium content. Histological preparations mainder
of the aorta were studied
for fat and general
5 mm) was from the re-
appearance.
Calcium analysis The three small sections of aorta were placed in test tubes where they were dried to a constant weight at 100 “C. After drying, the fat was extracted by washing the aortae in 10 ml portions of ethyl ether until the weights were constant again. The aortae were ashed at 600 “C until only a pure white ash remained. The tissue ash was dissolved in a 1% lanthanum chloride solution in 0.2 N hydrochloric acid, and the calcium content was determined in the solution with a Jarrell-Ash atomic absorption spectrophotometer. Histological methods The histological techniques were those described in detail by Lillieg. The three sections of aortae were further subdivided into sections approximately 5 mm in length and fixed in 10 % aqueous formaldehyde (U.S.P.). Complete cross sections were made through each section of the aortae. The hematoxylin and eosin method was used to show fibrosis
and aortic lesions.
RESULTS
Blood pressure Fig. 1 shows the systolic blood pressure of the four experimental groups rabbits from the beginning of the experiment until termination. At the beginning
x
of of
Normal
s Cholesterol o Cholesterol + Propronolol . Normal + Propranolol
80
0
I
2
3
4
WEEKS
5
6
7
8
9
IO
ON DIET
Fig. 1. Systolic blood pressures of rabbits on various experimental mean, not shown, ranged from 0.8 to 3.6 mm Hg.
diets. Standard
errors of the
340
P. J. WHITTINGTON-COLEMAN, 0. CARRlER, JR., B. H. DOUGLAS
the experiment there was no significant difference (P > 0.05) in the mean systolic blood pressure of any of the groups. After the fourth week there was a slight but not significant drop in blood pressure of the normal plus propranolol group. By the fifth week, both groups receiving the daily injections of propranolol had significantly lower (P < 0.05) blood pressures than the normal group or the 2 % cholesterol group. From the seventh week until the end of the experiment, there was no significant in the mean systolic blood pressure of any of the groups.
difference
Body weights The food consumption of all the animals at the beginning of the experiment approximately 75 g/day/rabbit. There was a gradual rise in food consumption,
was and
in the final weeks of the experiment the consumption rose to 150 g/day/rabbit. At no time in the experiment was there any significant difference in the food intake of any of the groups. The changes in the body weights of the four experimental groups were followed. There was a gradual increase in body weights of all groups as the 10 weeks of the experiment progressed. At no time in the experiment was there any signifidant difference at the 1 ‘A level in the weights of the four groups of rabbits. Tissue calcium Table 1 shows the calcium content of the vascular tissue of the four groups of experimental animals. The aortic calcium of the cholesterol fed group is significantly higher (P < 0.05) than propranolol.
that
of the normal
Serum cholesterol The results of the serum cholesterol TABLE CALCIUM
group
and
determinations
the two groups
receiving
are given in Table 2. There
1 CONTENT
OF AORTIC
TISSUE
OF RABBITS
ON VARIOUS
EXPERIMENTAL
DIETS
FOR
10
WEEKS
Diets consisted of normal Purina rabbit chow alone or with 2% by weight cholesterol added prior to pelleting (diets prepared by Nutritional Biochemicals). Group
Control
Diet
normal cholesterol cholesterol + 5.0 mg propranolol/kg/day normal diet + 5.0 mg propranolol/kg/day
Calcium (me&V./kg
10 10 8 8
1.41 9.56 7.55 7.32
f * & +
0.1” 0.8 0.30 0.6
P” dry weightc)
< 0.05 N.S. N.S.
Number of animals in groups. Significance of difference from normal group as determined by Student’s t-test (N.S. = not significant). Fat free. Mean value & S.E.M.
PROPRANOLOL
IN ATHEROMATOUS
341
LESIONS
TABLE 2 SERUM
CHOLESTEROL
CONTENT
OF RABBITS
ON VARIOUS
EXPERIMENTAL
DIETS
FOR
10
WEEKS
(for diets see Table 1) Group
Diet
N&
Serum cholesterolb (mg/lOO ml)
Control 1 2 3
normal cholesterol cholesterol + 5.0 mg propranolol/kg/day normal diet + 5.0 mg propranolol/kg/day
10 10 8 8
78.3 1103.6 2257 91
i 0.7 + 19.0 5 90 * 1.0
PC
< 0.01 < 0.01 < 0.05
& Number of animals in group. b At 10 weeks results are given as mean & S.E.M. c Significance of difference from the control group as determined by Student’s t-test.
was a sharp rise in serum cholesterol of the two groups receiving the 2 % cholesterol diet by the fifth week, and this level was maintained until the experiment was terminated. The serum cholesterol of the two groups receiving the 2 % cholesterol diet at the end of 10 weeks is significantly higher than that of the normal group or the group receiving a normal diet plus propranolol. At the end of the experiment the serum cholesterol level of the propranolol plus cholesterol group is significantly higher than that of the cholesterol Histological
group.
results
A normal rabbit aorta is shown in Fig. 2; there is no damage to the intima or the media of the vessel. After 10 weeks the group receiving the 2% cholesterol diet
Fig. 2. A normal rabbit aorta free of any vascular damage. Hematoxylin
and eosin, x 100.
342
P. J. WHITTINGTON-COLEMAN,
0. CARRIER,
JR., B. H. DOUGLAS
Fig. 3. Two per cent cholesterol group. The aorta has a grossly observable plaque attached to the intimal layer of the vessel. Hematoxylin and eosin, x 100. Fig. 4. Cholesterol and propranolol group. This nodule represents within this group. Hematoxylin and eosin stain, x 100.
the most advanced lesion seen
had large, grossly observable plaques in all sections of the aorta. This thick uniform band was attached to the intimal layer of the vessel and usually followed the entire circumference of the vessel (Fig. 3). The aortae from the group receiving the betaadrenergic blocker, propranolol, plus cholesterol had small atheromatous nodules on the intimal layer of the vessel in the arch area only. The nodule in Fig. 4 represents the most advanced lesion seen in this group. Lesions were not observed in any vessels from rabbits treated with propranolol plus a normal diet.
PROPRANOLOL
IN ATHEROMATOUS
343
LESIONS
DISCUSSION
In the present study we have shown that propranolol profoundly reduced the fat deposition along the intimal layer and inside the endothelial cells of the aortae while increasing serum cholesterol levels two-fold of rabbits receiving a 2% cholesterol diet. It also lowered the blood pressure
of both groups which received the drug
by the fifth week of the experiment. However after the seventh week, the blood pressure of both groups was back to normal and remained so throughout the experiment. It appears from this study that there may not be a direct relationship between the amount of fat deposited within the intimal layer of the aorta and the serum cholesterol levels. This suggests that some factor other than serum cholesterol level is responsible for the intimal development of the lesion. It has been reported that reserpine, guanethidine and two reserpine analogues prevent the development of atheromatous lesions in the aorta and reduce the calcium content of aortae in rabbits fed an atherogenic dieWe. It was suggested that these agents retarded or lessened the deposition of lipids in the aortic wall by a mechanism involving calcium depletion. A similar idea has also been proposed by Olwin and Koppelil. These authors demonstrated
that EDTA
which binds
calcium
reduced
the severity
of atherosclerosis
in
rabbits. In the present study, though propranolol did not reduce aortic calcium below control levels, it did prevent its increase during the development of the atheromatous lesions. Animals on the high cholesterol diet which received no propranolol had a significantly higher aortic calcium than did controls or those receiving propranolol. Calcification of vascular tissue initiated at some as yet unknown point in time, during the development of atherosclerotic disease, has long been known to occuria. The fact that the calcium content of atherosclerotic tissues progressively increases in such tissue has been well documented i3-17. Blumenthal and his co-worker+ have presented evidence that in the human, calcification of the aortic media commonly precedes plaque formation. Blankenhorn I3 feels that clacification of plaques begins as diffuse calcium deposition in the tissue before any signs of disease are apparent. The present results would suggest that if this early deposition of calcium (diffuse) is prevented then the lipid deposition will subsequently be prevented. Even though the calcium levels of the aortic tissue do appear to play an important role in the development of the atherosclerotic lesion the hypotensive action of propranolol cannot be disregarded at will. The hypotensive action of such drugs as reserpine, guanethidine and propranolol is certainly beneficial in the atherosclerotic process, for by lowering the pressure they prevent any undue physical stresses from further complicating the process, and hypertension does exacerbate the diseasels. It seems more likely, that the lessening and prevention of the atherosclerotic process observed in the present study arises from (I) the direct action of the pharmacologically active agents on some metabolic process involving calcium in the vascular wall and (2) that the hypotensive property of propranolol prevents further complication of the process. If such agents as reserpine, guanethidine and propranolol act to lower the bind-
P. J. WHLTTINGTON-COLEMAN,
344 ing
forces for calcium
at the cell membranes
0. CARRIER,
or the elastic
JR., B. H. DOUGLAS
tissue,
which has been
reported by Yu and Blumenthal20 to be the site of diffuse calcification, it is reasonable to assume that these agents may be preventing the deposition of lipid by such a mechanism. Studies with both the light microscope and the electron microscope have revealed that dense masses probably representing precipitated lipids are ous around elastic fiber&a2. Since elastic fibers are the site of diffuse and the site of the earliest lipid deposition, it is conceivable that calcium for lipid deposition. Thus, any agent which is able to prevent the diffuse should
be of benefit in inhibiting
the atherosclerotic
often numercalcification is necessary calcification
disease process.
REFERENCES 1 CARRIER,JR., O., CLOWER,B. R., AND WHITTINGTON,P. J., The inhibition of atheromatous lesions by reserpine, J. Afheroscler. Rex, 8 (1968) 229. 2 WHIT~INGTON-COLEMAN, P. J., CARRIER,JR., O., AND CLOWER, B. R., The effects of reserpine on vasopressin and cholesterol induced atheromatous lesions, J. Pharmacol. Exp. Therup., 160 (1968) 32. P., AND CARRIER, JR., O., Effects of agents altering vascular calcium in 3 WHITTINGTON-COLEMAN, 12 (1970) 15. experimental atherosclerosis, Atherosclerosis, 4 SCALES,B., AND MCINTOSH, D. A., Effects of propranolol and its optical isomers on the radio calcium uptake and the adenosin triphosphatases of skeletal and cardiac sarcoplasmic reticulum fractions, J. Pharmacol. Exp. Therup., 160 (1968) 261. 5 HESS,M. L., BRIGGS,F. N., SHINEBOURNE, E., AND HAMER J., Effect of adrenergic blocking agents on the calcium pump of the fragmented cardiac sarcoplasmic reticulum, Nature (LondonJ, 220 (1968) 79. 6 BOHR, D. F., Adrenergic receptors in coronary arteries, Ann. N.Y. Acad. Sci., 139 (1967) 799. I PRICHARD,B. N. C., AND GILLAM, P. M. S., Use of propranolol (Inderal) in treatment of hypertension, Brit. Med. J., ii (1964) 725. 8 PRICHARD, B. N. C., AND GILLAM, P. M. S., Treatment of hypertension with propranolol, &it. Med. J., i (1969) 7. 9 LILLIE, R. D., Histopathological Technic and Pwzcticul Histochemistry, McGraw-Hill, New York, 1965, p. 715. 10 WHITTINGTON,P. J., CLOWER, B. R., AND CARRIER, JR., O., Effects of reserpine on diet induced calcific atheromatous aortic lesions, Physiologist, 10 (1967) 343. 11 OLWIN, J. H., AND KOPPEL, J. L., Reduction of elevated plasma lipid levels in atherosclerosis following EDTA therapy, Proc. Sot. Exp. Biol. Med., 128 (1968) 1137. 12 KOLTZ, O., Studies upon calcareous degeneration, Part 1 (The process of pathological calcification), J. Exp. Med., 7 (1905) 633, 13 BLANKENHORN,D. H., Calcium deposits in the plaque. In: R. J. JONES(Ed.), Evolution 0,’the Atherosclerotic Plaque, The University of Chicago Press, Chicago, Ill., 1963, p. 297. 14 SERAFINI-FRACASSINI, A., AND GOTTE, L., EDTA decalcification of human mineralized aortas, J. Atheroscler. Res., 5 (1965) 61. 15 GARDNER, M. B., AND BLANKENHORN, D. H., Aortic medial calcification, Arch. Pathol., 85 (1968) 392. 16 BERKI, E., KORANYI, A., MAJOR, E., AND PERES,T., Ultrastructural study of inorganic substances in atherosclerotic aorta tissue, Cult. Tisszze Res., 4 (1969) 84. 17 STREBEL,R. F., AND WAGNER, B. M., Experimental tissue calcification, Part V (Effect of parathyroidectomy on spontaneously occurring calcific arteriosclerosis in female breeder rats), Arch. P&ho/., 87 (1969) 93. 18 BLUMENTHAL,H. T., LANSING,A. I., AND WHEELER,P. A., Calcification of the media of the human aorta and its relation to intimal arteriosclerosis, aging and disease, Amer. J. Puthol., 20 (1944) 665. 19 DEMING,Q. B., Hypertension and vascular disease. In: F. GROSS (Ed.), Antihypertensive Therapy, Springer, New York, 1966, p. 111.
PROPRANOLOL
20 Yu, S. Y.,
IN ATHEROMATOUS
LESIONS
345
ANDBLUMENTHAL, H. T., The calcification of elastic fiber, J. Atheroscler. Res., 5 (1965) 159. 21 CRAWFORD,T., Some aspects of the pathology of atherosclerosis, Proc. Roy. Sot. Med., 53 (1960) 9. 22 GEER, J. C., Fine structure of human aortic intimal thickening and fatty streaks, Lab. Invest., 14 (1965) 1764.
THE
EFFECTS
ROMATOUS
337 Amsterdam
OF PROPRANOLOL
- Printed in The Netherlands
ON CHOLESTEROL-INDUCED
ATHE-
LESIONS
PEGGY J. WHITTINGTON-COLEMAN*, GLAS*** Departments of Medicine and Pharmacology, 39216 (U.S.A.)
(Received November
OLIVER
CARRIER,
JR.**
AND
BEN H. DOU-
University of Mississippi Medical Center, Jackson, Miss.
14th, 1972)
SUMMARY
The effects of propranolol on cholesterol-induced atheromatous lesions in rabbits and on the calcium content of the vascular tissue of these animals were studied. The calcium content of normal rabbit aortae was significantly lower than that of the aortae of rabbits receiving a 2 % cholesterol supplemented diet. The calcium content from rabbit aortae which had received a 2% by weight cholesterol diet was significantly higher than that of the aortae of rabbits which received propranolol plus cholesterol, and of the aortae of rabbits which received a normal diet plus propranolol. Histological results showed atheromatous plaques within the intimal layer of the aortae of the cholesterol group. Some of the rabbits which received propranolol plus cholesterol had lesions, but these were less severe than those of the cholesterol group.
These results indicate
that propranolol
affects fat and calcium
metabolism
in
the aorta.
Key words :
Supported
Atheromatous
lesions
-
Propranolol - Serum
cholesterol
in patt by U.S. Public Health Service Grants HE-09031 and HE-09192. Postdoctoral Fellow and formerly NIH Predoctoral trainee (GM-39063). ** Present address: University of Texas at San Antonio, San Antonio, Texas, U.S.A. *** Established Investigator, American Heart Association. Please address all correspondence and requests for reprints to: Dr. Peggy J. WhittingtonColeman, Department of Medicine, University of Mississippi Medical Center, Jackson, Miss. 39216, U.S.A.
* Mississippi Heart Association
338
P. J. WHITTINGTON-COLEMAN,
0.
CARRIER,
JR.,
B. H. DOUGLAS
INTRODUCTION
Investigations in this laboratory have shown that cholesterol induced atherosclerosis in rabbits results in an increase in the total calcium and lipids of the sclerotic aortaeiy2. We have also shown that hypotensive agents such as reserpine, guanethidine and the reserpine
analogue,
methyl-0-reserpate
(SU-7064),
as well as the reserpine
analogue with no hypotensive properties (methyl-18-epireserpate methyl ether), prevent a rise in aortic calcium and the development of atherosclerotic lesions in rabbits fed a high cholesterol dietiJ. It has been demonstrated that propranolol depresses the sarco-tubular calcium pump4 in concentrations that depress cardiac contractilitys. Bohr6 has suggested that the beta-blockers decrease the amount of ionized calcium available to the cell. Prichard and Gillam7,s reported that propranolol produced a slight but significant degree of hypotension
in humans.
Since propranolol
apparently
affects both
calcium
meta-
bolism and has hypotensive properties it appeared conceivable that it could have a protective action against diet-induced atherosclerosis either by decreasing the blood pressure or by some mechanism involving calcium metabolism. The subject of the present report is our observations on the effect of propranolol on the development of the atherosclerotic lesion. METHODS
For this study 36 albino rabbits of either sex, weighing between 2.0 and 2.5 kg were used. Ten of the rabbits were maintained on a normal Purina diet throughout the experiment. Group 1 (10 animals)
received a normal
Purina
rabbit
pellet diet supplemented
with 2% cholesterol by weight. Group 2 (8 animals) received the same dietary regimen as Group 1, but were additionally treated with 5 mg of propranolol/kg/day (Inderal) administered intraperitoneally from the beginning of the experiment. Group 3 (8 animals) received a normal Purina rabbit pellet diet and were additionally treated with 5 mg of propranolol/kg/day administered intraperitoneally. Systolic blood pressures were recorded three times a week from an ear artery by a Grant-Rothschild capsule. The animals had ad libitum access to diet and water. Food consumption was followed throughout the experiment. Total body weight measurements were made weekly until the experiment was terminated. Blood samples were drawn from an ear artery at the beginning of the experiment, after 5 weeks and when the experiment terminated. Serum cholesterol levels were determined by a modified Lieberman-Buchard reaction. The animals were sacrificed by a sharp blow on the head 10 weeks after onset of the experimental feeding period. The aortae were removed from the aortic valve to the bifurcation. After the aortae had been removed from the animals they were cleaned of blood and divided into three sections. Section one consisted of the aortic
339
PROPRANOLOL IN ATHEROMATOUS LESIONS
arch from the aortic valve to the first pair of intercostal intercostal
arteries
to the coeliac artery
and section
arteries,
section two from the
three from the coeliac artery to
the bifurcation. The proximal portion of each section (approximately removed and analyzed for calcium content. Histological preparations mainder
of the aorta were studied
for fat and general
5 mm) was from the re-
appearance.
Calcium analysis The three small sections of aorta were placed in test tubes where they were dried to a constant weight at 100 “C. After drying, the fat was extracted by washing the aortae in 10 ml portions of ethyl ether until the weights were constant again. The aortae were ashed at 600 “C until only a pure white ash remained. The tissue ash was dissolved in a 1% lanthanum chloride solution in 0.2 N hydrochloric acid, and the calcium content was determined in the solution with a Jarrell-Ash atomic absorption spectrophotometer. Histological methods The histological techniques were those described in detail by Lillieg. The three sections of aortae were further subdivided into sections approximately 5 mm in length and fixed in 10 % aqueous formaldehyde (U.S.P.). Complete cross sections were made through each section of the aortae. The hematoxylin and eosin method was used to show fibrosis
and aortic lesions.
RESULTS
Blood pressure Fig. 1 shows the systolic blood pressure of the four experimental groups rabbits from the beginning of the experiment until termination. At the beginning
x
of of
Normal
s Cholesterol o Cholesterol + Propronolol . Normal + Propranolol
80
0
I
2
3
4
WEEKS
5
6
7
8
9
IO
ON DIET
Fig. 1. Systolic blood pressures of rabbits on various experimental mean, not shown, ranged from 0.8 to 3.6 mm Hg.
diets. Standard
errors of the
340
P. J. WHITTINGTON-COLEMAN, 0. CARRlER, JR., B. H. DOUGLAS
the experiment there was no significant difference (P > 0.05) in the mean systolic blood pressure of any of the groups. After the fourth week there was a slight but not significant drop in blood pressure of the normal plus propranolol group. By the fifth week, both groups receiving the daily injections of propranolol had significantly lower (P < 0.05) blood pressures than the normal group or the 2 % cholesterol group. From the seventh week until the end of the experiment, there was no significant in the mean systolic blood pressure of any of the groups.
difference
Body weights The food consumption of all the animals at the beginning of the experiment approximately 75 g/day/rabbit. There was a gradual rise in food consumption,
was and
in the final weeks of the experiment the consumption rose to 150 g/day/rabbit. At no time in the experiment was there any significant difference in the food intake of any of the groups. The changes in the body weights of the four experimental groups were followed. There was a gradual increase in body weights of all groups as the 10 weeks of the experiment progressed. At no time in the experiment was there any signifidant difference at the 1 ‘A level in the weights of the four groups of rabbits. Tissue calcium Table 1 shows the calcium content of the vascular tissue of the four groups of experimental animals. The aortic calcium of the cholesterol fed group is significantly higher (P < 0.05) than propranolol.
that
of the normal
Serum cholesterol The results of the serum cholesterol TABLE CALCIUM
group
and
determinations
the two groups
receiving
are given in Table 2. There
1 CONTENT
OF AORTIC
TISSUE
OF RABBITS
ON VARIOUS
EXPERIMENTAL
DIETS
FOR
10
WEEKS
Diets consisted of normal Purina rabbit chow alone or with 2% by weight cholesterol added prior to pelleting (diets prepared by Nutritional Biochemicals). Group
Control
Diet
normal cholesterol cholesterol + 5.0 mg propranolol/kg/day normal diet + 5.0 mg propranolol/kg/day
Calcium (me&V./kg
10 10 8 8
1.41 9.56 7.55 7.32
f * & +
0.1” 0.8 0.30 0.6
P” dry weightc)
< 0.05 N.S. N.S.
Number of animals in groups. Significance of difference from normal group as determined by Student’s t-test (N.S. = not significant). Fat free. Mean value & S.E.M.
PROPRANOLOL
IN ATHEROMATOUS
341
LESIONS
TABLE 2 SERUM
CHOLESTEROL
CONTENT
OF RABBITS
ON VARIOUS
EXPERIMENTAL
DIETS
FOR
10
WEEKS
(for diets see Table 1) Group
Diet
N&
Serum cholesterolb (mg/lOO ml)
Control 1 2 3
normal cholesterol cholesterol + 5.0 mg propranolol/kg/day normal diet + 5.0 mg propranolol/kg/day
10 10 8 8
78.3 1103.6 2257 91
i 0.7 + 19.0 5 90 * 1.0
PC
< 0.01 < 0.01 < 0.05
& Number of animals in group. b At 10 weeks results are given as mean & S.E.M. c Significance of difference from the control group as determined by Student’s t-test.
was a sharp rise in serum cholesterol of the two groups receiving the 2 % cholesterol diet by the fifth week, and this level was maintained until the experiment was terminated. The serum cholesterol of the two groups receiving the 2 % cholesterol diet at the end of 10 weeks is significantly higher than that of the normal group or the group receiving a normal diet plus propranolol. At the end of the experiment the serum cholesterol level of the propranolol plus cholesterol group is significantly higher than that of the cholesterol Histological
group.
results
A normal rabbit aorta is shown in Fig. 2; there is no damage to the intima or the media of the vessel. After 10 weeks the group receiving the 2% cholesterol diet
Fig. 2. A normal rabbit aorta free of any vascular damage. Hematoxylin
and eosin, x 100.
342
P. J. WHITTINGTON-COLEMAN,
0. CARRIER,
JR., B. H. DOUGLAS
Fig. 3. Two per cent cholesterol group. The aorta has a grossly observable plaque attached to the intimal layer of the vessel. Hematoxylin and eosin, x 100. Fig. 4. Cholesterol and propranolol group. This nodule represents within this group. Hematoxylin and eosin stain, x 100.
the most advanced lesion seen
had large, grossly observable plaques in all sections of the aorta. This thick uniform band was attached to the intimal layer of the vessel and usually followed the entire circumference of the vessel (Fig. 3). The aortae from the group receiving the betaadrenergic blocker, propranolol, plus cholesterol had small atheromatous nodules on the intimal layer of the vessel in the arch area only. The nodule in Fig. 4 represents the most advanced lesion seen in this group. Lesions were not observed in any vessels from rabbits treated with propranolol plus a normal diet.
PROPRANOLOL
IN ATHEROMATOUS
343
LESIONS
DISCUSSION
In the present study we have shown that propranolol profoundly reduced the fat deposition along the intimal layer and inside the endothelial cells of the aortae while increasing serum cholesterol levels two-fold of rabbits receiving a 2% cholesterol diet. It also lowered the blood pressure
of both groups which received the drug
by the fifth week of the experiment. However after the seventh week, the blood pressure of both groups was back to normal and remained so throughout the experiment. It appears from this study that there may not be a direct relationship between the amount of fat deposited within the intimal layer of the aorta and the serum cholesterol levels. This suggests that some factor other than serum cholesterol level is responsible for the intimal development of the lesion. It has been reported that reserpine, guanethidine and two reserpine analogues prevent the development of atheromatous lesions in the aorta and reduce the calcium content of aortae in rabbits fed an atherogenic dieWe. It was suggested that these agents retarded or lessened the deposition of lipids in the aortic wall by a mechanism involving calcium depletion. A similar idea has also been proposed by Olwin and Koppelil. These authors demonstrated
that EDTA
which binds
calcium
reduced
the severity
of atherosclerosis
in
rabbits. In the present study, though propranolol did not reduce aortic calcium below control levels, it did prevent its increase during the development of the atheromatous lesions. Animals on the high cholesterol diet which received no propranolol had a significantly higher aortic calcium than did controls or those receiving propranolol. Calcification of vascular tissue initiated at some as yet unknown point in time, during the development of atherosclerotic disease, has long been known to occuria. The fact that the calcium content of atherosclerotic tissues progressively increases in such tissue has been well documented i3-17. Blumenthal and his co-worker+ have presented evidence that in the human, calcification of the aortic media commonly precedes plaque formation. Blankenhorn I3 feels that clacification of plaques begins as diffuse calcium deposition in the tissue before any signs of disease are apparent. The present results would suggest that if this early deposition of calcium (diffuse) is prevented then the lipid deposition will subsequently be prevented. Even though the calcium levels of the aortic tissue do appear to play an important role in the development of the atherosclerotic lesion the hypotensive action of propranolol cannot be disregarded at will. The hypotensive action of such drugs as reserpine, guanethidine and propranolol is certainly beneficial in the atherosclerotic process, for by lowering the pressure they prevent any undue physical stresses from further complicating the process, and hypertension does exacerbate the diseasels. It seems more likely, that the lessening and prevention of the atherosclerotic process observed in the present study arises from (I) the direct action of the pharmacologically active agents on some metabolic process involving calcium in the vascular wall and (2) that the hypotensive property of propranolol prevents further complication of the process. If such agents as reserpine, guanethidine and propranolol act to lower the bind-
P. J. WHLTTINGTON-COLEMAN,
344 ing
forces for calcium
at the cell membranes
0. CARRIER,
or the elastic
JR., B. H. DOUGLAS
tissue,
which has been
reported by Yu and Blumenthal20 to be the site of diffuse calcification, it is reasonable to assume that these agents may be preventing the deposition of lipid by such a mechanism. Studies with both the light microscope and the electron microscope have revealed that dense masses probably representing precipitated lipids are ous around elastic fiber&a2. Since elastic fibers are the site of diffuse and the site of the earliest lipid deposition, it is conceivable that calcium for lipid deposition. Thus, any agent which is able to prevent the diffuse should
be of benefit in inhibiting
the atherosclerotic
often numercalcification is necessary calcification
disease process.
REFERENCES 1 CARRIER,JR., O., CLOWER,B. R., AND WHITTINGTON,P. J., The inhibition of atheromatous lesions by reserpine, J. Afheroscler. Rex, 8 (1968) 229. 2 WHIT~INGTON-COLEMAN, P. J., CARRIER,JR., O., AND CLOWER, B. R., The effects of reserpine on vasopressin and cholesterol induced atheromatous lesions, J. Pharmacol. Exp. Therup., 160 (1968) 32. P., AND CARRIER, JR., O., Effects of agents altering vascular calcium in 3 WHITTINGTON-COLEMAN, 12 (1970) 15. experimental atherosclerosis, Atherosclerosis, 4 SCALES,B., AND MCINTOSH, D. A., Effects of propranolol and its optical isomers on the radio calcium uptake and the adenosin triphosphatases of skeletal and cardiac sarcoplasmic reticulum fractions, J. Pharmacol. Exp. Therup., 160 (1968) 261. 5 HESS,M. L., BRIGGS,F. N., SHINEBOURNE, E., AND HAMER J., Effect of adrenergic blocking agents on the calcium pump of the fragmented cardiac sarcoplasmic reticulum, Nature (LondonJ, 220 (1968) 79. 6 BOHR, D. F., Adrenergic receptors in coronary arteries, Ann. N.Y. Acad. Sci., 139 (1967) 799. I PRICHARD,B. N. C., AND GILLAM, P. M. S., Use of propranolol (Inderal) in treatment of hypertension, Brit. Med. J., ii (1964) 725. 8 PRICHARD, B. N. C., AND GILLAM, P. M. S., Treatment of hypertension with propranolol, &it. Med. J., i (1969) 7. 9 LILLIE, R. D., Histopathological Technic and Pwzcticul Histochemistry, McGraw-Hill, New York, 1965, p. 715. 10 WHITTINGTON,P. J., CLOWER, B. R., AND CARRIER, JR., O., Effects of reserpine on diet induced calcific atheromatous aortic lesions, Physiologist, 10 (1967) 343. 11 OLWIN, J. H., AND KOPPEL, J. L., Reduction of elevated plasma lipid levels in atherosclerosis following EDTA therapy, Proc. Sot. Exp. Biol. Med., 128 (1968) 1137. 12 KOLTZ, O., Studies upon calcareous degeneration, Part 1 (The process of pathological calcification), J. Exp. Med., 7 (1905) 633, 13 BLANKENHORN,D. H., Calcium deposits in the plaque. In: R. J. JONES(Ed.), Evolution 0,’the Atherosclerotic Plaque, The University of Chicago Press, Chicago, Ill., 1963, p. 297. 14 SERAFINI-FRACASSINI, A., AND GOTTE, L., EDTA decalcification of human mineralized aortas, J. Atheroscler. Res., 5 (1965) 61. 15 GARDNER, M. B., AND BLANKENHORN, D. H., Aortic medial calcification, Arch. Pathol., 85 (1968) 392. 16 BERKI, E., KORANYI, A., MAJOR, E., AND PERES,T., Ultrastructural study of inorganic substances in atherosclerotic aorta tissue, Cult. Tisszze Res., 4 (1969) 84. 17 STREBEL,R. F., AND WAGNER, B. M., Experimental tissue calcification, Part V (Effect of parathyroidectomy on spontaneously occurring calcific arteriosclerosis in female breeder rats), Arch. P&ho/., 87 (1969) 93. 18 BLUMENTHAL,H. T., LANSING,A. I., AND WHEELER,P. A., Calcification of the media of the human aorta and its relation to intimal arteriosclerosis, aging and disease, Amer. J. Puthol., 20 (1944) 665. 19 DEMING,Q. B., Hypertension and vascular disease. In: F. GROSS (Ed.), Antihypertensive Therapy, Springer, New York, 1966, p. 111.
PROPRANOLOL
20 Yu, S. Y.,
IN ATHEROMATOUS
LESIONS
345
ANDBLUMENTHAL, H. T., The calcification of elastic fiber, J. Atheroscler. Res., 5 (1965) 159. 21 CRAWFORD,T., Some aspects of the pathology of atherosclerosis, Proc. Roy. Sot. Med., 53 (1960) 9. 22 GEER, J. C., Fine structure of human aortic intimal thickening and fatty streaks, Lab. Invest., 14 (1965) 1764.