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Inhibition of dietary atherosclerosis in rabbits by norethynodrel
Journal of A therosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
I N H I B I T I O N OF D I E T A R Y A T H E R O S C L E R O S I S IN R A B B I T S BY N O R E T H Y N O D R E L
I. GORE, Y. IWANAGA AND HAZEL GORE Department of Pathology, University Hospital, Boston University Medical Center, Boston, Mass. (U.S.A.)
(Received August 17th, 1966)
SUMMARY Norethynodrel administered to rabbits reduces the atherogenicity of a cholesterol diet in part through its effect on serum cholesterol. There is an increase of the aortic acid mucopolysaccharides of the treated animals, an observation which contradicts the theory that such accumulations predispose to the occurrence and localization of lipid deposits.
INTRODUCTION The observation that adult premenopausal women are less susceptible to atherosclerotic disease than similarly aged men 1 underlies the use of female hormones for its treatment and prevention 2. It has been established that estrogens have a distinct hypocholesterolemic effect 3. Evidence is also available indicating that estrogen therapy leads to improved survival in victims of coronary atherosclerosis 4,5. In chickens and rats, moreover, regression of experimentally induced coronary intimal lesions has resulted from the administration of estrogens 6. Because of these beneficial effects, little attention has been paid until recently to the influence of progesterone, another ovarian hormone which varies cyclically. MANALO-ESTRELLAand associates 7 described reversible structural alterations in the aortas of pregnant rabbits which could be duplicated by the administration of norethynodrel-mestranol. SPAIN AND MESTEL8, though unable to substantiate the presence of histologic alterations, observed that the preparation inhibited the development of atherosclerosis in cholesterol-fed rabbits. Unfortunately, the presence of the estrogen, mestranol, complicated interpretation of their findings. Norethynodrel or closely related steroids are the essential constituent of several Supported in parts by grants from NIH, HE-07327, and The Council for Tobacco Research, U.S.A. J. Atheroscler. Res., 7 (1967) 361-366
362
I. GORE, Y. IWANAGA, H. GORE
c o m m e r c i a l l y a v a i l a b l e c o n t r a c e p t i v e pills. T h e i r p o p u l a r i t y a n d w i d e s p r e a d usage w a r a n t s m o r e t h a n an a c a d e m i c i n t e r e s t in t h e i r r e p o r t e d effect upon b l o o d vessels. Accordingly, t h e studies o u t l i n e d below were c o n d u c t e d using the single agent nore t h y n o d r e l to: (i) establish w h e t h e r or n o t it i n h i b i t s t h e d e v e l o p m e n t of d i e t a r y atherosclerosis; (ii) d e t e r m i n e if a n d h o w it alters t h e s t r u c t u r e of t h e aortic wall; (iii) d e t e r m i n e , in t h e event t h a t changes are d e m o n s t r a b l e , how these m i g h t relate to t h e d e v e l o p m e n t of a t h e r o m a t o u s lesions. MATERIALS AND METHODS S e v e n t y y o u n g a d u l t female r a b b i t s weighing 1.4-2.0 kg were d i v i d e d into e x p e r i m e n t a l groups as i n d i c a t e d in Fig. 1. N o r e t h y n o d r e l s u s p e n d e d in m i n e r a l oil, was a d m i n i s t e r e d b y m o u t h at t h e r a t e of 2 m g / d a y to 43 r a b b i t s *. A t t h e end of t h r e e weeks 6 of t h e n o r e t h y n o d r e l t r e a t e d a n i m a l s were sacrificed. T r e a t m e n t was cont i n u e d an a d d i t i o n a l four weeks in 21 r a b b i t s , 13 of which were killed p r o m p t l y a n d t h e o t h e r s a f t e r a lapse of five clays (3) a n d nine d a y s (5), respectively. Sixteen r a b b i t s , p r e v i o u s l y t r e a t e d with n o r e t h y n o d r e l for t h r e e weeks were placed on a cholesterol reinforced d i e t for periods of four (5 rabbits) a n d six weeks (11 rabbits) d u r i n g which n o r e t h y n o d r e l t r e a t m e n t was continued. P a r a l l e l regimens were a d m i n i s t e r e d to 27 c o n t r o l animals. All were m a i n t a i n e d on a diet of P u r i n a r a b b i t chow for t h r e e weeks. T h r e e r a b b i t s were sacrificed a t this time, a n d 9 others c o n t i n u e d on stock diet for four a n d six a d d i t i o n a l weeks, respectively. Cholesterol was a d d e d to t h e diet for 15 c o n t r o l r a b b i t s , 5 for four weeks a n d 10 for six weeks.
NORETHYNODREL (43) 2 mg daily, p.o.
I
|
|
CONTROL(27)
II II II CHOLESTEROL DIET
I
II
I /
Fig. 1. Experimental plan (female rabbits, 1.4-2.0 kg). As depicted graphically, there were two groups of animals, a norethynodrel treated group and a control group. After three weeks of treatment priming of the first group, a portion of each was placed upon a cholesterol enriched diet for periods of 4-6 weeks. Each arrow indicates by its length, the period the listed number of rabbits were maintained before sacrifice. In the norethynodrel stock fed group, cross hatching of the arrows below the 4-week line represents a short period without hormonal treatment.
* The drug, free from other functionally active steroids, was generously provided by F. J. Saunders, Searle, Chicago, IlL j . Atheroscler. Res., 7 (1967) 361-366
INHIBITION OF DIETARY ATHEROSCLEROSIS IN RABBITS
363
Complete autopsies were performed upon all animals. Blood was removed from the left ventricle for the determination of cholesterol content. Atherosclerosis was assayed in the aorta b y visual estimation of the proportion of the surface affected. Representative portions of the vital organs and aortic wall were fixed in 1 % cetyl pyridinium chloride (CPC)--formalin (10 %) and processed through paraffin for histologic examination. The stains used consisted of hematoxylin and eosin, elastic tissue--Masson, and alcian blue--PAS. The remainder (and bulk) of the aortas were used for the quantitative extraction and fractionation of the acid mucopolysaccharides 9. OBSERVATIONS
As summarized in Fig. 2, rabbits receiving norethynodrel developed less aortic atherosclerosis on a cholesterol diet than untreated animals. Six of ten rabbits on the standard cholesterol diet for six weeks, had intimal lesions involving 20 % or more of the aortic surface, whereas, this was true of only one of the eleven norethynodrel treated animals. Two of the latter, moreover, displayed only minor involvement of the arterial surface in contrast to the uniform presence of substantial atherosclerosis in the untreated group. The mean percent (4- standard error) of the aortic intimal surface bearing lipid lesions (11.8 • 3.8) in the norethynodrel treated animals, was significantly lower (P < 0.05) than it was in the untreated cholesterol fed controls (27.2 4- 6.0). The coronary arteries were virtually unaffected in either group. Parallel
14000
I00
80
1~ooo~
o
i '~
.o[ No. of RobNts
ililll,.. 11,~176176 Cholesterol IO
Chol.-No~etllynodrr Ill
Fig. 2. R a b b i t s fed cholesterol for 6 w e e k s are c o m p a r e d w i t h a similar g r o u p receiving nore t h y n o d r e l . T h e vertical b a r s indicate t h e p e r c e n t of t h e aortic surface b e a r i n g lipid lesions in each rabbit. T h e t e r m i n a l s e r u m cholesterol v a l u e of each a n i m a l is i n d i c a t e d b y a s m a l l circle. T h e c o n n e c t i n g lines indicate t h e considerable i n d i v i d u a l v a r i a b i l i t y of t h i s s u b s t a n c e a n d t h e lack of c o r r e s p o n d e n c e w i t h t h e o b s e r v e d degree of atherosclerosis.
J. Atheroscler. Res., 7 (1967) 361-366
364
I. GORE, Y. IWANAGA, H. GORE
5,0"
4.9, 9
4J9
4.03
" ~ 4.0.
E
4.18 - -
~9.6 %
29.6 %
2~1
Z6,8
25.2
-~- 3.0" 0 o
2.0-
1.0-
Rob~fs
CS-C
25.7
NN 29.5
STOCK DiET NORETHYN. 12
CS-B
26.0 YHMS
28.1
r5.6
No. of
30.0
%
29.1 %
27
29.8
/15.41 ~
AHA
CHOL. DiET NORETHYN, 9
II
Fig. 3. The acid mucopolysaccharide (AMPS) c o n t e n t of the a o r t a indicating t h e effect of nore t h y n o d r e l t r e a t m e n t u p o n stock fed and cholesterol fed rabbits. The height of each c o l u m n represents t h e q u a n t i t y of AMPS isolated. The fractional composition is indicated b y t h e subdivisions into CS-C, chondroitin sulfate C; CS-B, chondroitin sulfate B; H M S h e p a r i t i n m o n o sulfate; a n d HA, hyaluronic acid. The a r r o w heads identify significant changes a n d indicate t h e direction of t h e change. The actual figures and the s t a n d a r d error are t a b u l a t e d in t h e accomp a n y i n g table.
differences (P < 0.05) were observed in the levels of serum cholesterol. The mean value (:k standard error) of this constituent in hormonally treated rabbits was 1556 ~: 155 mg/100 ml, whereas the mean control level was 2009 4- 374 mg/100 ml. Both groups were alike in regard to food intake (80-100 g of pellets per rabbit daily), weight gain, and physical activity. Histologically, except for the quantitative differences in the degree of intimal involvement, the aortas of treated and untreated animals were indistinguishable. However, the considerable enlargement of the uterus in norethynodrel treated rabbits testified to the effectiveness of hormone administration. Quantitative extraction of aortic acid mucopolysaccharides indicated no effect of norethynodrel on stock fed rabbits. On a cholesterol diet, however, the agent TABLE I NORETHYNODR]~L
AND AORTIC ACID MUCOPOLY'SACCHARIDES
Diet
Stock diet Stock diet with n o r e t h y nodrel Cholesterol diet Cholesterol diet w i t h norethynodrel
Uronic acid
(~g/g)
Fractions ( % )a HA
HMS
4.03 4- 0.42
14.4
-4- 1.82
4.19 4- 0.55 4.18 4- 0.62
15.6 4- 2.56 15.42 4- 1.39
29.5
CS-B • 2.56
28.1 • 3.37 29.79 :t: 2.23
27.1
CS-C •
1.56
26.8 -4- 3.25 25.17 4- 2.63
29.1
:J: 1.63
29.6 4- 1.03 29.62 4- 1.03
4.91 4- 0.57 b 18.21 4- 1.40 b 26.06 4- 1.28 b 25.74 4- 1.61 b 30.03 • 2.54
a See Fig. 3. b p < 0.02 or better.
j . Atheroscler. Res., 7 (1967) 361-366
INHIBITION OF DIETARY ATHEROSCLEROSIS IN RABBITS
365
produced a s u b s t a n t i a l increase characterized b y relative a n d absolute increases of h y a l u r o n i c acid a n d a relative reduction of heparitin sulfate. The other c o n s t i t u e n t s of aortic acid mucopolysaccharides, chondroitin sulfates B a n d C, were unaffected (Fig. 3, Table I). DISCUSSION
The e x p e r i m e n t a l findings indicate t h a t n o r e t h y n o d r e l t r e a t m e n t reduces the atherogenicity of a cholesterol diet for r a b b i t s a n d t h a t this effect is associated with lowered serum values of cholesterol. I t does n o t seem likely t h a t the slight estrogenic a c t i v i t y of n o r e t h y n o d r e l 1~ is the e x p l a n a t i o n , since in rabbits, estrogens do n o t alter the arterial response to cholesterol feeding n . Altered susceptibility of the vessel wall m a y be inferred from reports of n o r e t h y n o d r e l i n d u c e d increases of aortic acid mucopolysaccharides 7, indeed, our own findings are confirmatory, b u t only with cholesterol s u p p l e m e n t a t i o n of the diet. I t is p e r t i n e n t to c o m m e n t , however, t h a t a u g m e n t e d aortic deposits of acid mucopolysaccharides (AMPS) in this e x p e r i m e n t at least, were associated with diminished susceptibility to the d e v e l o p m e n t of i n t i m a l lesions c o n t r a r y to a widely accepted hypothesis of atherogenesis 12-14. P e r t i n e n t to this direct contradiction, it is to be n o t e d t h a t other hormones which influence e x p e r i m e n t a l atherosclerosis have no consistent effect on the AMPS c o n t e n t of aortic tissue. R e d u c e d atherosclerosis from t r e a t m e n t with estrogenl, 15 or cortisone 16-1s is associated with reduced aortic AMPS 1~, b u t these substances are u n changed b y t h y r o x i n t r e a t m e n t 20 which enhances aortic i n t i m a l disease in rabbits~0, ~1, a n d fails to ameliorate coronary atherogenesis in cholesterol fed cockerels 22. As yet, it is n o t possible to assign significance to the compositional differences observed in the arterial AMPS of the t r e a t e d animals.
REFERENCES 1 ]~ERKSON,D. M., J. STAMLERAND D. B. COHEN,Ovarian function and coronary atherosclerosis, Clin. Obstet. Gynecol., 1964, 7: 504. 2 OLIVER, M. F. AND G. S. BOYD, Thyroid and estrogen treatment of hypercholesterolemia in man. In G. PINCUS(Ed.), Hormones and A therosclerosis, Academic Press, New York, 1959, p. 423. a EDER, H. A., The effects of sex hormones on serum lipids and lipoproteins. In G. PINCUS(Ed)., Hormones and Atherosclerosis, Academic Press, New York, 1959. 4 STAMLER, J., R. PICK, L. N. KATZ, A. PICK, B. KAPLAN, D. BERKSON AND D. CENTURY, Effectiveness of estrogens for therapy of myocardial infarction in middle age men, J. Am. Med. Assoc., 1963, 183: 632. 5 •ARMORSTON, J., F. J. MOORE, O. T. KUZMA,O. MAGIDSONAND J. WEINER, Survival benefit of estrogen in men with myocardial infarction, Clin. Res., 1961, 9: 62. 6 PICK, R., J. STAMLERAND L. N. KATZ, Influence of estrogens on lipids and atherosclerosis in experimental animals. In G. PINCUS (Ed.), Hormones and Atherosclerosis, Academic Press, New York, p. 423. 7 MANALO-ESTRELLA, P., C. t3. TAYLORAND A. K. ASDEL, Effects of pregnancy and hormone treatment on arterial structure and atherogenesis in rabbits, Circulation, 1965, 32: Suppl. II : 11-2. s SPAIN, D. M. AND A. L. MESTEL, Influence of norethynodrel with mestranol on atherogenesis in rabbits, Circulation, 1965, 32: Suppl. II : 1I 32. j . Atheroscler. Res., 7 (1967) 361-366
366
I. GORE, Y. IWANAGA, H. GORE
9 TANAKA, V. AND I. GORE, Cellulose column c h r o m a t o g r a p h y for the fractionation a n d isolation of acid mucopolysaccharides, ]. Chromatog., 1966, 23: 254. t0 SAUNDERS, F. J., R. A. EDGREN AND V. A. ]:)RILL, On t h e progestational activity of ITAethynyl-17 hydroxy-5 (10)-estren-3-one (norethynodrel), Endocrinology, 1957, 60: 804. 11 STAMLER, J., R. PICK AND L. N. KATZ, Experiences in assessing estrogen anti-atherogenesis in t h e chick, t h e rabbit, and man, Ann. N. Y. Acad. Sci., 1956, 64: 596. 12 GER6, S., Investigations on t h e role of vascular mucopolysaccharides in t h e m e c h a n i s m of lipid deposition, Symp. Zool. Soc. London, 1964, I1: 169. 13 MOON, H. D. AND J. F. RINEHART, Histogenesis of coronary atherosclerosis, Circulation, 1952, 6: 481. 14 MILLER. B. F., F. P. KEYES AND R. W. CURRERI, Increase of serum fl-glucuronidase in h u m a n diabetes mellitus, J. Am. Med. Assoc., 1966, 195: 189. 15 MOSKOWITZ, M. S., A. A. MOSKOWITZ, W. L. BRADFORD, JR. AND R. W. WISSLER, Changes in t h e s e r u m lipids a n d coronary arteries of t h e r a t in response to estrogens, Arch. Pathol., 1956, 61: 245. 16 MALINOW, M. R., R. DEPAOLI, C. A. MARUFFO, J. STEVENS, I. SZlJAN AND S. J. KAPLAN, T h e effect of estradiol and hydrocortisone on atherosclerosis in cockerels, J. Atheroscler. Res., 1965, 5:403. 17 ADLERSBERG, D., Adrenocortical hormones a n d experimental atherosclerosis. I n G. PINCUS (Ed.), Hormones and Atherosclerosis, Academic Press, New York, 1959, p. 484. 18 FRIEDMAN, M., S. BYERS AND S. S. ST. GEORGE, Cortisone and experimental atherosclerosis, Arch. Pathol., 1964.77: 142. 19 PRIEST, R. E., R. M. KOPLITZ AND E. P. BENDITT, Estradiol reduces incorporation of radioactive sulfate into cartilage a n d aortas of rats, J. Exptl. Med., 1960, 112: 225. 20 GORE, I., Y. IWANAGA, Y. TANAKA AND M. L. GOODMAN, Thyroid hormone a n d atherogenesis (Proc. Am. Assoc. Pathol. Bacteriol.), Am. ]. Pathol., 1966, 48: 34A, No. 6. 21 FISHER, E. R., Thyroidal influence on experimental cholesterol atherosclerosis, A m. J. Pathol., 1964, 45: 21. 32 JAIN, S., R. PICK, P. J. JOHNSON AND L. N. KATZ, Failure of D- a n d L-thyroxine to protect cholesterol a n d oil fed cockerels against coronary atherogenesis, Circulation Res., 1966, 18: 519.
J. Atheroscler. Res., 7 (1967) 361-366
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
I N H I B I T I O N OF D I E T A R Y A T H E R O S C L E R O S I S IN R A B B I T S BY N O R E T H Y N O D R E L
I. GORE, Y. IWANAGA AND HAZEL GORE Department of Pathology, University Hospital, Boston University Medical Center, Boston, Mass. (U.S.A.)
(Received August 17th, 1966)
SUMMARY Norethynodrel administered to rabbits reduces the atherogenicity of a cholesterol diet in part through its effect on serum cholesterol. There is an increase of the aortic acid mucopolysaccharides of the treated animals, an observation which contradicts the theory that such accumulations predispose to the occurrence and localization of lipid deposits.
INTRODUCTION The observation that adult premenopausal women are less susceptible to atherosclerotic disease than similarly aged men 1 underlies the use of female hormones for its treatment and prevention 2. It has been established that estrogens have a distinct hypocholesterolemic effect 3. Evidence is also available indicating that estrogen therapy leads to improved survival in victims of coronary atherosclerosis 4,5. In chickens and rats, moreover, regression of experimentally induced coronary intimal lesions has resulted from the administration of estrogens 6. Because of these beneficial effects, little attention has been paid until recently to the influence of progesterone, another ovarian hormone which varies cyclically. MANALO-ESTRELLAand associates 7 described reversible structural alterations in the aortas of pregnant rabbits which could be duplicated by the administration of norethynodrel-mestranol. SPAIN AND MESTEL8, though unable to substantiate the presence of histologic alterations, observed that the preparation inhibited the development of atherosclerosis in cholesterol-fed rabbits. Unfortunately, the presence of the estrogen, mestranol, complicated interpretation of their findings. Norethynodrel or closely related steroids are the essential constituent of several Supported in parts by grants from NIH, HE-07327, and The Council for Tobacco Research, U.S.A. J. Atheroscler. Res., 7 (1967) 361-366
362
I. GORE, Y. IWANAGA, H. GORE
c o m m e r c i a l l y a v a i l a b l e c o n t r a c e p t i v e pills. T h e i r p o p u l a r i t y a n d w i d e s p r e a d usage w a r a n t s m o r e t h a n an a c a d e m i c i n t e r e s t in t h e i r r e p o r t e d effect upon b l o o d vessels. Accordingly, t h e studies o u t l i n e d below were c o n d u c t e d using the single agent nore t h y n o d r e l to: (i) establish w h e t h e r or n o t it i n h i b i t s t h e d e v e l o p m e n t of d i e t a r y atherosclerosis; (ii) d e t e r m i n e if a n d h o w it alters t h e s t r u c t u r e of t h e aortic wall; (iii) d e t e r m i n e , in t h e event t h a t changes are d e m o n s t r a b l e , how these m i g h t relate to t h e d e v e l o p m e n t of a t h e r o m a t o u s lesions. MATERIALS AND METHODS S e v e n t y y o u n g a d u l t female r a b b i t s weighing 1.4-2.0 kg were d i v i d e d into e x p e r i m e n t a l groups as i n d i c a t e d in Fig. 1. N o r e t h y n o d r e l s u s p e n d e d in m i n e r a l oil, was a d m i n i s t e r e d b y m o u t h at t h e r a t e of 2 m g / d a y to 43 r a b b i t s *. A t t h e end of t h r e e weeks 6 of t h e n o r e t h y n o d r e l t r e a t e d a n i m a l s were sacrificed. T r e a t m e n t was cont i n u e d an a d d i t i o n a l four weeks in 21 r a b b i t s , 13 of which were killed p r o m p t l y a n d t h e o t h e r s a f t e r a lapse of five clays (3) a n d nine d a y s (5), respectively. Sixteen r a b b i t s , p r e v i o u s l y t r e a t e d with n o r e t h y n o d r e l for t h r e e weeks were placed on a cholesterol reinforced d i e t for periods of four (5 rabbits) a n d six weeks (11 rabbits) d u r i n g which n o r e t h y n o d r e l t r e a t m e n t was continued. P a r a l l e l regimens were a d m i n i s t e r e d to 27 c o n t r o l animals. All were m a i n t a i n e d on a diet of P u r i n a r a b b i t chow for t h r e e weeks. T h r e e r a b b i t s were sacrificed a t this time, a n d 9 others c o n t i n u e d on stock diet for four a n d six a d d i t i o n a l weeks, respectively. Cholesterol was a d d e d to t h e diet for 15 c o n t r o l r a b b i t s , 5 for four weeks a n d 10 for six weeks.
NORETHYNODREL (43) 2 mg daily, p.o.
I
|
|
CONTROL(27)
II II II CHOLESTEROL DIET
I
II
I /
Fig. 1. Experimental plan (female rabbits, 1.4-2.0 kg). As depicted graphically, there were two groups of animals, a norethynodrel treated group and a control group. After three weeks of treatment priming of the first group, a portion of each was placed upon a cholesterol enriched diet for periods of 4-6 weeks. Each arrow indicates by its length, the period the listed number of rabbits were maintained before sacrifice. In the norethynodrel stock fed group, cross hatching of the arrows below the 4-week line represents a short period without hormonal treatment.
* The drug, free from other functionally active steroids, was generously provided by F. J. Saunders, Searle, Chicago, IlL j . Atheroscler. Res., 7 (1967) 361-366
INHIBITION OF DIETARY ATHEROSCLEROSIS IN RABBITS
363
Complete autopsies were performed upon all animals. Blood was removed from the left ventricle for the determination of cholesterol content. Atherosclerosis was assayed in the aorta b y visual estimation of the proportion of the surface affected. Representative portions of the vital organs and aortic wall were fixed in 1 % cetyl pyridinium chloride (CPC)--formalin (10 %) and processed through paraffin for histologic examination. The stains used consisted of hematoxylin and eosin, elastic tissue--Masson, and alcian blue--PAS. The remainder (and bulk) of the aortas were used for the quantitative extraction and fractionation of the acid mucopolysaccharides 9. OBSERVATIONS
As summarized in Fig. 2, rabbits receiving norethynodrel developed less aortic atherosclerosis on a cholesterol diet than untreated animals. Six of ten rabbits on the standard cholesterol diet for six weeks, had intimal lesions involving 20 % or more of the aortic surface, whereas, this was true of only one of the eleven norethynodrel treated animals. Two of the latter, moreover, displayed only minor involvement of the arterial surface in contrast to the uniform presence of substantial atherosclerosis in the untreated group. The mean percent (4- standard error) of the aortic intimal surface bearing lipid lesions (11.8 • 3.8) in the norethynodrel treated animals, was significantly lower (P < 0.05) than it was in the untreated cholesterol fed controls (27.2 4- 6.0). The coronary arteries were virtually unaffected in either group. Parallel
14000
I00
80
1~ooo~
o
i '~
.o[ No. of RobNts
ililll,.. 11,~176176 Cholesterol IO
Chol.-No~etllynodrr Ill
Fig. 2. R a b b i t s fed cholesterol for 6 w e e k s are c o m p a r e d w i t h a similar g r o u p receiving nore t h y n o d r e l . T h e vertical b a r s indicate t h e p e r c e n t of t h e aortic surface b e a r i n g lipid lesions in each rabbit. T h e t e r m i n a l s e r u m cholesterol v a l u e of each a n i m a l is i n d i c a t e d b y a s m a l l circle. T h e c o n n e c t i n g lines indicate t h e considerable i n d i v i d u a l v a r i a b i l i t y of t h i s s u b s t a n c e a n d t h e lack of c o r r e s p o n d e n c e w i t h t h e o b s e r v e d degree of atherosclerosis.
J. Atheroscler. Res., 7 (1967) 361-366
364
I. GORE, Y. IWANAGA, H. GORE
5,0"
4.9, 9
4J9
4.03
" ~ 4.0.
E
4.18 - -
~9.6 %
29.6 %
2~1
Z6,8
25.2
-~- 3.0" 0 o
2.0-
1.0-
Rob~fs
CS-C
25.7
NN 29.5
STOCK DiET NORETHYN. 12
CS-B
26.0 YHMS
28.1
r5.6
No. of
30.0
%
29.1 %
27
29.8
/15.41 ~
AHA
CHOL. DiET NORETHYN, 9
II
Fig. 3. The acid mucopolysaccharide (AMPS) c o n t e n t of the a o r t a indicating t h e effect of nore t h y n o d r e l t r e a t m e n t u p o n stock fed and cholesterol fed rabbits. The height of each c o l u m n represents t h e q u a n t i t y of AMPS isolated. The fractional composition is indicated b y t h e subdivisions into CS-C, chondroitin sulfate C; CS-B, chondroitin sulfate B; H M S h e p a r i t i n m o n o sulfate; a n d HA, hyaluronic acid. The a r r o w heads identify significant changes a n d indicate t h e direction of t h e change. The actual figures and the s t a n d a r d error are t a b u l a t e d in t h e accomp a n y i n g table.
differences (P < 0.05) were observed in the levels of serum cholesterol. The mean value (:k standard error) of this constituent in hormonally treated rabbits was 1556 ~: 155 mg/100 ml, whereas the mean control level was 2009 4- 374 mg/100 ml. Both groups were alike in regard to food intake (80-100 g of pellets per rabbit daily), weight gain, and physical activity. Histologically, except for the quantitative differences in the degree of intimal involvement, the aortas of treated and untreated animals were indistinguishable. However, the considerable enlargement of the uterus in norethynodrel treated rabbits testified to the effectiveness of hormone administration. Quantitative extraction of aortic acid mucopolysaccharides indicated no effect of norethynodrel on stock fed rabbits. On a cholesterol diet, however, the agent TABLE I NORETHYNODR]~L
AND AORTIC ACID MUCOPOLY'SACCHARIDES
Diet
Stock diet Stock diet with n o r e t h y nodrel Cholesterol diet Cholesterol diet w i t h norethynodrel
Uronic acid
(~g/g)
Fractions ( % )a HA
HMS
4.03 4- 0.42
14.4
-4- 1.82
4.19 4- 0.55 4.18 4- 0.62
15.6 4- 2.56 15.42 4- 1.39
29.5
CS-B • 2.56
28.1 • 3.37 29.79 :t: 2.23
27.1
CS-C •
1.56
26.8 -4- 3.25 25.17 4- 2.63
29.1
:J: 1.63
29.6 4- 1.03 29.62 4- 1.03
4.91 4- 0.57 b 18.21 4- 1.40 b 26.06 4- 1.28 b 25.74 4- 1.61 b 30.03 • 2.54
a See Fig. 3. b p < 0.02 or better.
j . Atheroscler. Res., 7 (1967) 361-366
INHIBITION OF DIETARY ATHEROSCLEROSIS IN RABBITS
365
produced a s u b s t a n t i a l increase characterized b y relative a n d absolute increases of h y a l u r o n i c acid a n d a relative reduction of heparitin sulfate. The other c o n s t i t u e n t s of aortic acid mucopolysaccharides, chondroitin sulfates B a n d C, were unaffected (Fig. 3, Table I). DISCUSSION
The e x p e r i m e n t a l findings indicate t h a t n o r e t h y n o d r e l t r e a t m e n t reduces the atherogenicity of a cholesterol diet for r a b b i t s a n d t h a t this effect is associated with lowered serum values of cholesterol. I t does n o t seem likely t h a t the slight estrogenic a c t i v i t y of n o r e t h y n o d r e l 1~ is the e x p l a n a t i o n , since in rabbits, estrogens do n o t alter the arterial response to cholesterol feeding n . Altered susceptibility of the vessel wall m a y be inferred from reports of n o r e t h y n o d r e l i n d u c e d increases of aortic acid mucopolysaccharides 7, indeed, our own findings are confirmatory, b u t only with cholesterol s u p p l e m e n t a t i o n of the diet. I t is p e r t i n e n t to c o m m e n t , however, t h a t a u g m e n t e d aortic deposits of acid mucopolysaccharides (AMPS) in this e x p e r i m e n t at least, were associated with diminished susceptibility to the d e v e l o p m e n t of i n t i m a l lesions c o n t r a r y to a widely accepted hypothesis of atherogenesis 12-14. P e r t i n e n t to this direct contradiction, it is to be n o t e d t h a t other hormones which influence e x p e r i m e n t a l atherosclerosis have no consistent effect on the AMPS c o n t e n t of aortic tissue. R e d u c e d atherosclerosis from t r e a t m e n t with estrogenl, 15 or cortisone 16-1s is associated with reduced aortic AMPS 1~, b u t these substances are u n changed b y t h y r o x i n t r e a t m e n t 20 which enhances aortic i n t i m a l disease in rabbits~0, ~1, a n d fails to ameliorate coronary atherogenesis in cholesterol fed cockerels 22. As yet, it is n o t possible to assign significance to the compositional differences observed in the arterial AMPS of the t r e a t e d animals.
REFERENCES 1 ]~ERKSON,D. M., J. STAMLERAND D. B. COHEN,Ovarian function and coronary atherosclerosis, Clin. Obstet. Gynecol., 1964, 7: 504. 2 OLIVER, M. F. AND G. S. BOYD, Thyroid and estrogen treatment of hypercholesterolemia in man. In G. PINCUS(Ed.), Hormones and A therosclerosis, Academic Press, New York, 1959, p. 423. a EDER, H. A., The effects of sex hormones on serum lipids and lipoproteins. In G. PINCUS(Ed)., Hormones and Atherosclerosis, Academic Press, New York, 1959. 4 STAMLER, J., R. PICK, L. N. KATZ, A. PICK, B. KAPLAN, D. BERKSON AND D. CENTURY, Effectiveness of estrogens for therapy of myocardial infarction in middle age men, J. Am. Med. Assoc., 1963, 183: 632. 5 •ARMORSTON, J., F. J. MOORE, O. T. KUZMA,O. MAGIDSONAND J. WEINER, Survival benefit of estrogen in men with myocardial infarction, Clin. Res., 1961, 9: 62. 6 PICK, R., J. STAMLERAND L. N. KATZ, Influence of estrogens on lipids and atherosclerosis in experimental animals. In G. PINCUS (Ed.), Hormones and Atherosclerosis, Academic Press, New York, p. 423. 7 MANALO-ESTRELLA, P., C. t3. TAYLORAND A. K. ASDEL, Effects of pregnancy and hormone treatment on arterial structure and atherogenesis in rabbits, Circulation, 1965, 32: Suppl. II : 11-2. s SPAIN, D. M. AND A. L. MESTEL, Influence of norethynodrel with mestranol on atherogenesis in rabbits, Circulation, 1965, 32: Suppl. II : 1I 32. j . Atheroscler. Res., 7 (1967) 361-366
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9 TANAKA, V. AND I. GORE, Cellulose column c h r o m a t o g r a p h y for the fractionation a n d isolation of acid mucopolysaccharides, ]. Chromatog., 1966, 23: 254. t0 SAUNDERS, F. J., R. A. EDGREN AND V. A. ]:)RILL, On t h e progestational activity of ITAethynyl-17 hydroxy-5 (10)-estren-3-one (norethynodrel), Endocrinology, 1957, 60: 804. 11 STAMLER, J., R. PICK AND L. N. KATZ, Experiences in assessing estrogen anti-atherogenesis in t h e chick, t h e rabbit, and man, Ann. N. Y. Acad. Sci., 1956, 64: 596. 12 GER6, S., Investigations on t h e role of vascular mucopolysaccharides in t h e m e c h a n i s m of lipid deposition, Symp. Zool. Soc. London, 1964, I1: 169. 13 MOON, H. D. AND J. F. RINEHART, Histogenesis of coronary atherosclerosis, Circulation, 1952, 6: 481. 14 MILLER. B. F., F. P. KEYES AND R. W. CURRERI, Increase of serum fl-glucuronidase in h u m a n diabetes mellitus, J. Am. Med. Assoc., 1966, 195: 189. 15 MOSKOWITZ, M. S., A. A. MOSKOWITZ, W. L. BRADFORD, JR. AND R. W. WISSLER, Changes in t h e s e r u m lipids a n d coronary arteries of t h e r a t in response to estrogens, Arch. Pathol., 1956, 61: 245. 16 MALINOW, M. R., R. DEPAOLI, C. A. MARUFFO, J. STEVENS, I. SZlJAN AND S. J. KAPLAN, T h e effect of estradiol and hydrocortisone on atherosclerosis in cockerels, J. Atheroscler. Res., 1965, 5:403. 17 ADLERSBERG, D., Adrenocortical hormones a n d experimental atherosclerosis. I n G. PINCUS (Ed.), Hormones and Atherosclerosis, Academic Press, New York, 1959, p. 484. 18 FRIEDMAN, M., S. BYERS AND S. S. ST. GEORGE, Cortisone and experimental atherosclerosis, Arch. Pathol., 1964.77: 142. 19 PRIEST, R. E., R. M. KOPLITZ AND E. P. BENDITT, Estradiol reduces incorporation of radioactive sulfate into cartilage a n d aortas of rats, J. Exptl. Med., 1960, 112: 225. 20 GORE, I., Y. IWANAGA, Y. TANAKA AND M. L. GOODMAN, Thyroid hormone a n d atherogenesis (Proc. Am. Assoc. Pathol. Bacteriol.), Am. ]. Pathol., 1966, 48: 34A, No. 6. 21 FISHER, E. R., Thyroidal influence on experimental cholesterol atherosclerosis, A m. J. Pathol., 1964, 45: 21. 32 JAIN, S., R. PICK, P. J. JOHNSON AND L. N. KATZ, Failure of D- a n d L-thyroxine to protect cholesterol a n d oil fed cockerels against coronary atherogenesis, Circulation Res., 1966, 18: 519.
J. Atheroscler. Res., 7 (1967) 361-366