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Experimental atherosclerosis as observed in the chicken
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REVIEW E X P E R I M E N T A L A T H E R O S C L E R O S I S AS O B S E R V E D IN T H E C H I C K E N L. N. KATZ AND R. PICK Cardiovascular Department, Medical Research Institute, Michael Reese Hospital and Medical Center, Chicago, Ill. ( U . S . A . )
(Received August 27th, 1960) INTRODUCTION ANITSCHKOW1 in 1913 was the first investigator to produce true atherosclerosis in an experimental animal (the rabbit) b y feeding cholesterol. From 1913 to the late 1930's research in this area had become fairly d o r m a n t because of adverse criticism a b o u t using a herbivorous animal to which a dietary constituent alien to the species was given. Since then, these objections have been overwhelmingly proven to be in error. Many workers have repeated Anitschkow's experiments and his original findings have been extended to numerous other animal species. In all these experiments, cholesterol-fat feeding has been the s i n e q u a n o n for the production of atherosclerosis. In some species these dietary measures had to be combined with other regimens, either hormonal (in the dog) or dietary (in rats and monkeys), to m a k e t h e m effective 9. The only m a j o r exception to this rule is the production of aortic atherosclerosis in chicks on an ordinarily non-atherogenic diet b y estrogen-induced, endogenous hyperlipemia and hypercholesterolemia 9. This d e p a r t m e n t has been actively engaged in atherosclerosis research since 1940 16,18. After a careful survey of the literature existing at t h a t time the chick was selected as the experimental animal and has been used by the d e p a r t m e n t in the last decade and a half. This avian species spontaneously develops a gross arterial lesion similar to h u m a n atherosclerosis 30. In addition, as was early d e m o n s t r a t e d in the initial experiments, it responds to cholesterol-oil feeding b y developing gross atherosclerosis of all the m a j o r arteries 2,4-9,15,21,28,29. Moreover, the production of atherosclerotic aortic lesions b y an endogenously produced hyperlipemia and hypercholesterolemia afforded the s t u d y of a third type of atherosclerotic lesion 31. The main morphologic features of chick atherosclerosis are as follows: (1) The spontaneous lesions in the intima of the aorta and the coronary arteries are almost certainly primarily a fibrotic process, i.e., an arteriosclerotic (not atherosclerotic) lesion. Cholesterol feeding and estrogen exhibition aggravate and intensify the spontaneous aortic lesions. I t would, therefore, appear t h a t the fibrotic spontaneous plaque is a site of predilection for cholesterol and lipid deposition, with subsequent evolutionary atherosclerotic changesT,9, 30. (2) The p r i m a r y lesion of both experimental cholesterol and estrogen-induced atherosclerosis is the foam cell intimal cushion. This lesion, the so-called pure atheJ. Atheroscler. Res., 1 (1961) 93-100
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roma, is almost certainly the first stage of experimental atherogenesis. It is not preceded by mucoprotein deposition7,9, ao. (3) The morphologic patterns of more advanced lesions are apparently the result of pathologic processes secondary to atheroma formation and continued lipid deposition. B y prolonged cholesterol feeding the spectrum of atherosclerotic changes as seen in human lesions, including foam cell plaques, necrosis and atheromatous abscesses, fibrosis and hyalinization, calcification and cartilagenous-osseous metaplasia, can be producedT,9,30. (4) Another feature, common in human atherosclerosis but heretofore not produced in an experimental animal, namely ulceration, has also been produced recently in the chick61, 62. This will be discussed below. A. D I E T A R Y S T U D I E S
Early in these studies, precise quantitation between cholesterol-intake, hypercholesterolemia and atherogenesis was achievedg,39, 40. B y this means, it was possible to demonstrate that atherosclerosis can be produced with minimal hypercholesterolemia and organ lipidosis provided a sufficiently long time is allowed for their productiong, 3s. These experiments once more prove the similarity of experimental atherosclerosis to the happenings in human beings a. The quick production of lesions with excessive hypercholesterolemia and organ lipidosis is, therefore, only an expedient, not a compulsory part of experimental work 34. More recent experiments in cholesterol-induced atherosclerosis has brought to the fore the question of the interrelationship of different dietary factors in experimental atherosclerosis 1~ Thus, it was shown that the protein content of the diet in the presence of a cholesterol-oil supplement is decisive for the magnitude of the atherosclerotic vascular involvement achieved. A high protein diet will retard while a low protein diet will accelerate atherosclerotic vascular involvement at a given cholesterol-oil supplement2~,52,53,56, 57. It was also shown that the type of carbohydrate used in an atherogenic diet will determine its atherogenicity. This has prompted the expression of a "dietary balance" hypothesis for atherosclerosis19,23. It has also been shown that the early atherosclerotic lesions of coronary arteries in the chick are readily reversible in 2-3 weeks by eliminating the dietary cholesteroloil supplement and reverting to an unsupplemented normal mash diet 35. Aortic lesions take longer to regress. An interrelationship between protein content and reversibility of lesions was also shown to exist during the regression period. However, the interrelationship during regression is opposite in nature to the relationship found during the induction of lesions, that is to say a high protein supplement during the regression period will retard regression of lesions while a low protein diet will accelerate regression63. The mechanism of these interrelationships is not clear and is at present the subject of intensive investigation. There is also some indication that certain vitamin deficiencies m a y be related to the development of atherosclerosis in the presence of a potentially atherogenic diet. J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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These interrelationships however are so complex that much more work has to be done before they can be clarified. In more recent studies, it was also demonstrated that the mode of ingestion of the atherogenic diet influences the development and regression of vascular lesions. This was shown in short-term experiments and, therefore, is most clearly demonstrated in the behavior of the p a t t e r n of hypercholesterolemia and in the development and regression of coronary lesions 60. Chicks trained to eat their food in two periods of one hour each ("meal eaters") developed more severe hypercholesterolemia and atherosclerosis t h a n did chicks eating the same quantity continuously all day long ("nibblers"). Also, when regular mash was substituted after a period of cholesterol-oil feeding, the a t h e r o m a t o u s lesions regressed faster when the regular mash diet was nibbled than when this diet was given as meals. From these dietary experiments it can be concluded t h a t the chick is an excellent experimental animal for studies on the influence of diet on atherosclerosis, and it is hoped that these experiments can continue to give insight into the dietary influences playing a role in the pathogenesis of human atherosclerosis. B. HORMONALINFLUENCES
I t has long been known t h a t hormonal influences play a role in the development of atherosclerosis in animals on a potentially atherogenic dietll,14,22,26,2L I t has been shown t h a t thyroid hormone will decrease the atherogenicity of a given cholesterol-oil supplemented diet while hypothyroidism increases its atherogenicity. In dogs, atherosclerosis can only be produced b y combining a high-cholesterol, high-fat diet with suppression of thyroid activity either b y thiouracil feeding, surgical t h y roidectomy, or 131I administration. Adrenal medullary and cortical hormones h a v e also been shown to influence the development of atherosclerosis in several animal species. The hormones of the pancreas have a distinct effect on atherosclerosis. Of particular interest, for several reasons, is the effect of the sex hormones on coronary atherogenesis. (I) The thyroid. T h y r o i d hormone given to chicks on an atherogenic diet, either as desiccated thyroid powder or thyroxin, suppresses hypercholesterolemia and, usually but not consistently in all experiments, also decreases atherogenesisg,26,33,36,aT, 54. The inconsistent effects on atherogenesis m a y be due to the additional action of thyroid hormone in causing vascular damage and, therefore, predisposing to increased a t h e r o m a formation at mild hypercholesterolemic levels. Furthermore, it was found t h a t thyroid medication does not accelerate regression of previously induced atherosclerosis 26. Hypothyroidism produced b y thiouracil administration consistently increases hypercholesterolemia and atherogenesis 9,28. Also, thiouracil-induced hypothyroidism partially counteracts the estrogen-induced protection of the coronary arteries, discussed below 51. (2) Adrenal steroids. Adrenal steroids show an effect on atherogenesis in chicks. The J. Atheroscler. Res., 1 (1961) 93-100
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gluco-corticoid hydrocortisone (compound F), the active adrenal steroid in the chick, produces enhanced hypercholesterolemia without affecting atherosclerosisU-13, 26, 27,47. This same effect was also demonstrated in the rabbit and most other animal species. The mineralo-corticoid desoxycorticosterone acetate (DCA) produces a slight hypertension in chicks and intensifies atherogenesis through this mechanism. When hypertension does not develop, no increase in severity of the vascular lesions is noted 11-13,26,41.
(3) Pancreas. Pancreatectomy is readily achieved in the chick, but pancreatectomized animals appear to behave to all intents and purposes like normal animals. T h e y do not show spontaneous hyperlipemia or hypercholesterolemia and they have normal blood-glucose levels and a normal glucose tolerance. However, on feeding cholesterol a subtle derangement of cholesterol metabolism can be shown. When cholesterol is given at a low percentage of the diet (0.25%) together with a low oil supplement, hypereholesterolemia and atherogenesis are similar in these pancreatectomized animals to those in the controls, but if a large (2%) cholesterol supplement and 5 % cottonseed oil are used, these pancreatectomized animals show enhanced hypercholesterolemia and atherosclerosis of the a o r t a and the coronary arteries 11,12,26,~7,42. As far as glucose metabolism is concerned, here too a subtle defect can be shown when these depancreatized animals are given c o m p o u n d F. Pancreatectomized animals respond to compound F with a three-fold increase in blood glucose as c o m p a r e d with normal hydrocortisone-treated animals. However, if hydrocortisone administration is combined with cholesterol feeding no e n h a n c e m e n t of atherogenesis can be producedn,13,42. These findings of a subtle disturbance of c a r b o h y d r a t e metabolism suggested an experimental analysis of the effects of insulin. I t was found t h a t insulin administered to normal non-diabetic animals on a cholesterol-oil supplemented, atherogenic diet does not influence atherogenesis. However, if insulin is given during the period of regression, after cholesterol feeding has been stopped, the regression of the atherosclerotic lesions is suppressed. This happens despite a reversal to normal of the dietinduced hypercholesterolemia. Preliminary experiments into the mechanisms of these insulin effects seem to indicate that hypoglycemia as such is involved in this phenomenon26, 59. However, more experiments with different hypoglycemia-producing agents will h a v e to be carried out to clarify this mechanism further. (d) Sex hormones. The hormones that show the most m a r k e d effect on experimental atherosclerosis in chicks are the estrogens20,25, 27. When cholesterol and estrogens are given concurrently, coronary atherogenesis is m a r k e d l y inhibited, despite the fact t h a t cholesterol-induced aorta atherogenesis proceeds unaffected4a, 5s. Moreover, it was also shown t h a t estrogen administration reverses coronary lesions previously induced b y cholesterol feeding, even when the cholesterol feeding is maintained during the period of estrogen exhibition44,49, 50. Here again, estrogen reversal of cholesterol-induced coronary atherosclerosis is associated with no such effect on aorta atherosclerotic lesions, provided the high-fat, high-cholesterol diet has a high protein content. J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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These experiments on estrogens and cholesterol-induced coronary atherosclerosis are of significance for several reasons. First, the plasma lipid d a t a recorded in these studies suggest t h a t the plasma ratio of cholesterol to phospholipids (C/P ratio) m a y be a significant factor in coronary atherogenesis. Thus, in all birds protected from coronary atherogenesis b y estrogen exhibition, plasma C/P ratios are at or near normal levels, in contrast to the control chicks fed cholesterol without estrogens, wherein C/P ratios are uniformly elevated. Further, the results of these experiments afford a lead to possible mechanisms in the well-known h u m a n sex-differential in susceptibility to coronary atherosclerosis 24. Finally, the results of these investigations highlight an i m p o r t a n t lesson for atherosclerosis research. So far, most investigators of experimental atherosclerosis had assumed t h a t the process of atherogenesis in different arterial beds is based upon essentially similar biological laws. Therefore, most workers confined their studies to the aorta and interpreted their findings there as applicable to atherogenesis in the coronary, renal, cerebral and other arteries. The results of these experiments on estrogens and coronary and aorta atherogenesis d e m o n s t r a t e beyond d o u b t t h a t this is a fundamental methodologic error. Since estrogen exhibition inhibits coronary atherogenesis in cholesterol-fed cockerels without exerting a n y effect on aorta atherogenesis, it m a y be concluded that different laws determine atherogenesis in the aorta and coronary arteries. Estrogens do not influence aortic atherosclerosis in chicks in which lesions were induced b y a high-cholesterol, high-fat, high-protein diet. However, if lesions were induced in animals b y a high-cholesterol, high-fat, low-protein diet and estrogens in large dosage are then exhibited, ulceration of aortic, particularly abdominal aortic, and coronary lesions were observedSX, 62. Thus, for the first time in experimental atherosclerosis, one other stage of atherosclerotic "complication" has been produced in the chicken. These experiments also indicated t h a t the ulceration of lesions in the animal m a y occur during the healing phase of atherosclerosis. This was shown b y carrying out serial weekly autopsies over a five week period. Preliminary d a t a indicate t h a t the tendency to ulceration is associated with definite morphologic differences between the appearance of the atherosclerotic lesion produced b y a high-protein diet and t h a t caused b y a low-protein diet. In the high-protein lesions a b u n d a n t fibrous connective tissue is laid down while the framework of the low-protein lesion is characterized b y a negligible fibroblastic activity. These data are being evaluated further. Estrogens continue to exert their protective action on coronary atherosclerosis in the chick even when combined with testosterone, adrenal hormones and pancreatectomy~5,45,4s, 55. The only regimen counteracting their effect is, as mentioned above, thiouracil-induced hypothyroidism26, 51. These findings seem to indicate t h a t a normal thyroid function is essential for the protective action of the estrogenic hormones on coronary atherosclerosis. CONCLUSION
This brief review of the main developments in experimental atherosclerosis in the chicken provides ample justification for using this animal species in experiments J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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attempting to analyze the pathogenesis and therapeutic value of different procedures and substances prior to their trial in man. It is, of course, clear beyond doubt that the last proof of the efficacy of any suggested regimen in man must ultimately rest on careful clinical trials. Animal experiments, in this regard, are merely a screening device. I t is also evident that the study of experimental atherosclerosis, and of the exogenous and endogenous factors influencing it, can yield knowledge of fundamental significance for the understanding of human atherosclerosis. Present knowledge does not permit us to state that the atherosclerosis problem has been solved by research along these lines. Much remains to be done. But it can be stated that considerable progress has been made in recent years since the epoch-making studies of ANITSCHKOW, and that this approach to the problem m a y ultimately bring the solution of the atherosclerosis problem, and with it the prevention, control and eventual elimination of this disease. ACKNOWLEDGEMENTS This research program of the department was made possible by generous support over the years by many private individuals and groups, and b y foundations and voluntary health agencies, as well as by grants from the Public Health Service of the United States Governement. The major sources of support included: The Michael Reese Research Foundation, the Lasker Foundation, The Chicago Heart Association, The American Heart Association and the National Heart Institute. Over the years a large group of physicians participated in the atherosclerosis research program. Among these, we wish to acknowledge particularly the most important contributions of Drs. D. V. DAUBER*, L. HORLICK, S. RODBARD AND J. STAMLER. These studies would not have been possible without the intelligent and skillful cooperation of the research assistants and technicians of the atherosclerosis research team. Especially deserving of mention are Mrs. C. BOLENE-WILLIAMS, Mrs. D. CENTURY AND Mr. P. JOHNSON, Research Assistants; and Miss C. KAKITA, Mrs. E. MILLER, Mrs. C. THOMPSON, Mrs. M. TRAVERS, Mrs. M. VANKINSCOTTAND Mr. G. CROWLEY,Technicians. SUMMARY The main morphologic features of spontaneous, cholesterol-induced and estrogeninduced atherosclerotic lesions in the chicken are described. The results of studies of dietary and hormonal influences on the formation and regression of these lesions are outlined. It is concluded that the results j astify the use of this animal species in the study of the pathogenesis of atherosclerosis and of the therapeutic value of different procedures and substances prior to their trial in man. * Deceased. J. Atheroscler. Res., 1 (1961) 93 100
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RESUM~
Les auteurs d6crivent les principales caract~ristiques morphologiques de ldsions ath6roscl6rotiques spontan6es provoqu6es chez le poussin par le cholest6rol et l'oestrog~ne. Ils exposent les r6sultats d'6tudes portant sur les influences di6t6tiques et hormonales sur la formation et la r6gression de ces 16sions. On conclut que les r6sultats justifient l'usage de cette espfice d'animaux pour 6tudier la pathogen&se de l'atMroscl6rose et la valeur thdrapeutique de diff6rentes m~thodes et substances avant de les 6prouver chez l'homme. ZUSAMMENFASSUNG
Die wichtigsten morphologischen Eigenttimlichkeiten der spontanen, bzw. durch Cholesterin oder Ostrogen hervorgerufenen krankhaften atherosklerotischen Ver/inderungen beim Kticken werden beschrieben. Die Ergebnisse der Untersuchungen tiber den Einfluss von Dffit und Hormonen auf die Entwicklung oder Rtickbildung dieser Ver~nderungen werden berichtet. Auf Grund dieser Ergebnisse erscheint das Kticken geeignet ffir das Studium der Pathogenese der Atherosklerose und des therapeutischen "vVertes yon verschiedenen Massnahmen und Substanzen vor ihrer Erprobung beim Menschen. REFERENCES E x c e p t for r e f e r e n c e to t h e r e v i e w b y ANITSCHKO~r, all o t h e r r e f e r e n c e s are confined to t h o s e p u b l i s h e d b y t h e d e p a r t m e n t . T h e w o r k of o t h e r s is referred to in t h e s e reports. T h e r e f e r e n c e s are divided into reviews, original contributions and preliminary notes, each arranged chronologically. 1 N. AmTSCHKOW, in E. V. C o w n R v , Arteriosclerosis, MacMillan, N e w York, t933, p. 271. Reviews 2 L . N . KATZ AND D. V. DAUBER, J. Mt. Sinai Hospital, 12 (1945) 382. 3 L . N . KATZ, J. SrAMLER AND L. HORLICK, Am. Practitioner, 1 (1950) 461. 4 L. N. KATZ, Trans. 5th Conference on Factors Regulating Blood Pressure, J o s i a h M a c y J r . F o u n d a t i o n , N e w Y o r k , 1951, p. 174. 5 L . N . KATZ, Circulation, 5 (t952) 101. 6 L. N. KATZ, J. STAMLER AND R. PICK, in Year Book Path. and Clin. Path., Chicago, 1951, p. 125 7 L. N. KATZ, J. STAMLER, R. PICK AND S. RODBARD, Lancet, 72 (1952) 326 ( P a r t I), 372 ( P a r t II). 8 L . N . KATZ, H. L. BLUMGART, Clinical Progress in Cardiovascular Disease, G r u n e a n d S t r a t t o n , N e w York, 1952, p. 9. 9 L, N. KATZ AND J. STAMLER, Experimental Atherosclerosis, C h a r l e s C. T h o m a s , Springfield, Ill., 1953. 10 L . N . KATZ, R . PICK, AND J. S'rAMLER, 2Plodern Concepts of Cardiovascular Disease, 23 (1954) 239. ll J. STAMLER, L. N. KATZ AND R. PICK, in H. SELYE AND G. HEUSER, dth Annual Report on Stress, A c t a Inc., M o n t r e a l , C a n a d a , 1954, p. 245. 1~ L . N . KATZ, J. STAMLER, aND R. PICK, Symposium on Atherosclerosis, N a t i o n a l A c a d e m y of Sciences, N a t i o n a l R e s e a r c h Council, W a s h i n g t o n , D.C., N . A . S . - N . R . C . P u b l i c a t i o n , 338 (1954) 236. x, j. STAMLER, L. N. I~ATZ, R . PICK AND S. RODBARD, Recent Progress in Hormone Research, Vol. 11, A c a d e m i c P r e s s Inc., N e w York, 1955, p. 4 0 t . 14 L . N . KATZ, A n n . Int. Med., 43 (1955) 930. 15 L. N. KATZ, in A. KEYS, Arteriosclerosis, a Symposium presented by the Minnesota Heart Association and the University of Minnesota, 1956, p. 25.
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L . N . KATZ, Circulation Research, 4 (1956) 123. L . N . KATZ, J. STAPLER AND R. PICK, J. A m . iVied. Assoc., 161 (1956) 536. L . N . KATZ, J. STAPLER AND R. PICK, Lancet, 76 (1956) 355. L . N . KATZ, J. STAPLER AND R. PICK, Federation Proc., 15 (1956) 885. J. STAPLER, R. PICK AND L. N. KATZ, A n n . N . Y . Acad. Sci., 64 (1956) 596. 21 L . N . KATZ, Symposium on Normal Limits and Functional Variations of the E CG. Proc. 3rd World Congress of Cardiology, Brussels, 1958, p. 403. 22 L . N . KATZ, J. STAPLER AND R. PICK, Festschrift Volume honoring $cademico Josef Charvat o n his 60th Birthday, S t a t e Medical Publishers, P r a g u e , 1958, p. 89. 23 L . N . KATZ, J. STAPLER AND R. PICK, Nutrition and Atherosclerosis, L e a a n d Febiger, P h i l a d e l p h i a , 1958. 24 J. STAPLER, R. PICK, L. N. KATZ, A. PICK AND B. M. KAPLAN, i n G. PINCUS, Hormones and Atherosclerosis, Academic Press, New York, 1959, C h a p t . 30, p. 423. 25 R. PICK, J. STAPLER AND L. 1kT. I~IATZ, in G. PINCUS, Hormones and A therosclerosis, A c a d e m i c Press, N e w York, 1959, Chapt. 18, p. 229. 26 J. STAPLER, R. PICK AND L. N. KATZ, in G. PINCUS, Hormones and Atherosclerosis, A c a d e m i c Press, N e w York, 1959, Chapt. 14, p. 173. 27 L . N . KATZ AND R. PICK, in H. T. BLUMENTHAL, The Hormonal Regulation of L i p i d Metabolism and its Etiological Significance, Charles C. T h o m a s , Springfield, II1., Chapt. 18, in t h e press. 16
17 18 19 20
Original Contributions 28 29
D . V . DAUBER AND L. N. KATZ, A..Vi.A. Arch. Pathol., 34 (1942) 937. D . V . DAUBER AND L. N. KATZ, A . M . A . Arch. Pathol., 36 (1943) 473.
30 31 32 33 34 35
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D . V . DAUBER, A . 3 I . A . Arch. Pathol., 38 (1944) 46. L. HORLICK AND L. N. KATZ, J. Lab. Clin. Med., 33 (1948) 733. L. HORLICK, L. N. KATZ AND J. STAMLER, A m . Heart J., 37 (1949) 689. D . V . DAUBER, L. HORLICK AND L. N. KATZ, A m . Heart J., 38 (1949) 25. L. HORLICK AND L. N. KATZ, Am. Heart J., 38 (1949) 336. L. HORLICK AND L. N. KATZ, J. Lab. Clin. Med., 34 (1949) 1427. J. STAPLER, E. N. SILBER, A. J. MILLER, L. AKMAN, C. BOLENE AND L. N. KATZ, J. Lab. Clin. 2Vied., 35 (1950) 351. J. STAPLER, A. J. MILLER, L. AKMAN, E. N. SILBER, C. BOLENE AND L. N. KATE, Circulation, 2 (1950) 523. J. STAPLER AND L. N. KATZ, Circulation, 2 (1950) 705. S. RODBARD, L. ~'q-. KATZ, C. BOLENE, R. PICK, M. LOWENTHAL AND G. GROS, Circulation,
40 41 42 43 44 45 46
3 (1951) 867. S. RODBARD, C. BOLENE AND L. N. KATZ, Circulation, 4 (1951) 43. J. STAPLER, R. PICK AND L. N. KATZ, Circulation, 4 (1951) 262. J. STAPLER AND L. N. KATZ, Circulation, 4 (1951) 255. R. PICK, J. STAPLER, S. RODBARD AND L. ~N~.KATZ, Circulation, 6 (1952) 276. 1~. PICK, J. STAPLER, S. RODBARD AND L. N. KATZ, Circulation, 6 (1952) 858. J. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 1 (1953) 94. S. RODBARD, C. BOLENE-V~TILLIAMS, R. PICK AND L. 1~. KATZ, J. Lab. Clin. Med., 41 (1953)
s6
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587. STAPLER, 1~. PICK AND L. N. KATZ, Circulation, 10 (1954) 237. STAPLER, R. PICK AND L. N. KATZ, Circulation, 10 (1954) 247. STAPLER, R. PICK AND L. N. KATZ, Circulation, 10 (1954) 251. PICK, J. STAMLER AND L. N. KATZ, Circulation Research, 5 (1957) 515. PICK, J. STAPLER AND L. N. KATZ, Circulation Research, 5 (1957) 510. STAMLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 442. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 447. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 825. PICK, J. STAPLER, S. RODBARD AND L. N. KATZ, Circulation Research, 7 (1959) 202. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 7 (1959) 398. PICK, J. STAMLER AND L. N. KATZ, Circulation Research, 7 (1959) 866. HOJMAN, A. A. PELLEGRINO-IRALDI, M. R. MALINOW, l:{. PICK J. STAPLER AND L. N.
47 48 49 50 51 52 53 54 55 56 57 5s
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KATZ, A.,Vi.A. Arch. Pathol., 68 (1959) 533. J. STAMLER, R. PICK AND L. N. KATZ, Circulation Research, 8 (1960) 572. C. COHN, R. PICK AND L. N. KATZ, Circulation Research, i n t h e press.
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Preliminary Notes 61 62 63
R. PICK, J. STAPLER AND L. N. KATZ, Federation Proc., 17 (1958) 124. R. PICK AND L. N. KATZ, Circulation, 18 (1958) 481. R. PICK AND L. N. KATZ, Federation Proc., 18 (1959) 120.
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REVIEW E X P E R I M E N T A L A T H E R O S C L E R O S I S AS O B S E R V E D IN T H E C H I C K E N L. N. KATZ AND R. PICK Cardiovascular Department, Medical Research Institute, Michael Reese Hospital and Medical Center, Chicago, Ill. ( U . S . A . )
(Received August 27th, 1960) INTRODUCTION ANITSCHKOW1 in 1913 was the first investigator to produce true atherosclerosis in an experimental animal (the rabbit) b y feeding cholesterol. From 1913 to the late 1930's research in this area had become fairly d o r m a n t because of adverse criticism a b o u t using a herbivorous animal to which a dietary constituent alien to the species was given. Since then, these objections have been overwhelmingly proven to be in error. Many workers have repeated Anitschkow's experiments and his original findings have been extended to numerous other animal species. In all these experiments, cholesterol-fat feeding has been the s i n e q u a n o n for the production of atherosclerosis. In some species these dietary measures had to be combined with other regimens, either hormonal (in the dog) or dietary (in rats and monkeys), to m a k e t h e m effective 9. The only m a j o r exception to this rule is the production of aortic atherosclerosis in chicks on an ordinarily non-atherogenic diet b y estrogen-induced, endogenous hyperlipemia and hypercholesterolemia 9. This d e p a r t m e n t has been actively engaged in atherosclerosis research since 1940 16,18. After a careful survey of the literature existing at t h a t time the chick was selected as the experimental animal and has been used by the d e p a r t m e n t in the last decade and a half. This avian species spontaneously develops a gross arterial lesion similar to h u m a n atherosclerosis 30. In addition, as was early d e m o n s t r a t e d in the initial experiments, it responds to cholesterol-oil feeding b y developing gross atherosclerosis of all the m a j o r arteries 2,4-9,15,21,28,29. Moreover, the production of atherosclerotic aortic lesions b y an endogenously produced hyperlipemia and hypercholesterolemia afforded the s t u d y of a third type of atherosclerotic lesion 31. The main morphologic features of chick atherosclerosis are as follows: (1) The spontaneous lesions in the intima of the aorta and the coronary arteries are almost certainly primarily a fibrotic process, i.e., an arteriosclerotic (not atherosclerotic) lesion. Cholesterol feeding and estrogen exhibition aggravate and intensify the spontaneous aortic lesions. I t would, therefore, appear t h a t the fibrotic spontaneous plaque is a site of predilection for cholesterol and lipid deposition, with subsequent evolutionary atherosclerotic changesT,9, 30. (2) The p r i m a r y lesion of both experimental cholesterol and estrogen-induced atherosclerosis is the foam cell intimal cushion. This lesion, the so-called pure atheJ. Atheroscler. Res., 1 (1961) 93-100
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roma, is almost certainly the first stage of experimental atherogenesis. It is not preceded by mucoprotein deposition7,9, ao. (3) The morphologic patterns of more advanced lesions are apparently the result of pathologic processes secondary to atheroma formation and continued lipid deposition. B y prolonged cholesterol feeding the spectrum of atherosclerotic changes as seen in human lesions, including foam cell plaques, necrosis and atheromatous abscesses, fibrosis and hyalinization, calcification and cartilagenous-osseous metaplasia, can be producedT,9,30. (4) Another feature, common in human atherosclerosis but heretofore not produced in an experimental animal, namely ulceration, has also been produced recently in the chick61, 62. This will be discussed below. A. D I E T A R Y S T U D I E S
Early in these studies, precise quantitation between cholesterol-intake, hypercholesterolemia and atherogenesis was achievedg,39, 40. B y this means, it was possible to demonstrate that atherosclerosis can be produced with minimal hypercholesterolemia and organ lipidosis provided a sufficiently long time is allowed for their productiong, 3s. These experiments once more prove the similarity of experimental atherosclerosis to the happenings in human beings a. The quick production of lesions with excessive hypercholesterolemia and organ lipidosis is, therefore, only an expedient, not a compulsory part of experimental work 34. More recent experiments in cholesterol-induced atherosclerosis has brought to the fore the question of the interrelationship of different dietary factors in experimental atherosclerosis 1~ Thus, it was shown that the protein content of the diet in the presence of a cholesterol-oil supplement is decisive for the magnitude of the atherosclerotic vascular involvement achieved. A high protein diet will retard while a low protein diet will accelerate atherosclerotic vascular involvement at a given cholesterol-oil supplement2~,52,53,56, 57. It was also shown that the type of carbohydrate used in an atherogenic diet will determine its atherogenicity. This has prompted the expression of a "dietary balance" hypothesis for atherosclerosis19,23. It has also been shown that the early atherosclerotic lesions of coronary arteries in the chick are readily reversible in 2-3 weeks by eliminating the dietary cholesteroloil supplement and reverting to an unsupplemented normal mash diet 35. Aortic lesions take longer to regress. An interrelationship between protein content and reversibility of lesions was also shown to exist during the regression period. However, the interrelationship during regression is opposite in nature to the relationship found during the induction of lesions, that is to say a high protein supplement during the regression period will retard regression of lesions while a low protein diet will accelerate regression63. The mechanism of these interrelationships is not clear and is at present the subject of intensive investigation. There is also some indication that certain vitamin deficiencies m a y be related to the development of atherosclerosis in the presence of a potentially atherogenic diet. J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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These interrelationships however are so complex that much more work has to be done before they can be clarified. In more recent studies, it was also demonstrated that the mode of ingestion of the atherogenic diet influences the development and regression of vascular lesions. This was shown in short-term experiments and, therefore, is most clearly demonstrated in the behavior of the p a t t e r n of hypercholesterolemia and in the development and regression of coronary lesions 60. Chicks trained to eat their food in two periods of one hour each ("meal eaters") developed more severe hypercholesterolemia and atherosclerosis t h a n did chicks eating the same quantity continuously all day long ("nibblers"). Also, when regular mash was substituted after a period of cholesterol-oil feeding, the a t h e r o m a t o u s lesions regressed faster when the regular mash diet was nibbled than when this diet was given as meals. From these dietary experiments it can be concluded t h a t the chick is an excellent experimental animal for studies on the influence of diet on atherosclerosis, and it is hoped that these experiments can continue to give insight into the dietary influences playing a role in the pathogenesis of human atherosclerosis. B. HORMONALINFLUENCES
I t has long been known t h a t hormonal influences play a role in the development of atherosclerosis in animals on a potentially atherogenic dietll,14,22,26,2L I t has been shown t h a t thyroid hormone will decrease the atherogenicity of a given cholesterol-oil supplemented diet while hypothyroidism increases its atherogenicity. In dogs, atherosclerosis can only be produced b y combining a high-cholesterol, high-fat diet with suppression of thyroid activity either b y thiouracil feeding, surgical t h y roidectomy, or 131I administration. Adrenal medullary and cortical hormones h a v e also been shown to influence the development of atherosclerosis in several animal species. The hormones of the pancreas have a distinct effect on atherosclerosis. Of particular interest, for several reasons, is the effect of the sex hormones on coronary atherogenesis. (I) The thyroid. T h y r o i d hormone given to chicks on an atherogenic diet, either as desiccated thyroid powder or thyroxin, suppresses hypercholesterolemia and, usually but not consistently in all experiments, also decreases atherogenesisg,26,33,36,aT, 54. The inconsistent effects on atherogenesis m a y be due to the additional action of thyroid hormone in causing vascular damage and, therefore, predisposing to increased a t h e r o m a formation at mild hypercholesterolemic levels. Furthermore, it was found t h a t thyroid medication does not accelerate regression of previously induced atherosclerosis 26. Hypothyroidism produced b y thiouracil administration consistently increases hypercholesterolemia and atherogenesis 9,28. Also, thiouracil-induced hypothyroidism partially counteracts the estrogen-induced protection of the coronary arteries, discussed below 51. (2) Adrenal steroids. Adrenal steroids show an effect on atherogenesis in chicks. The J. Atheroscler. Res., 1 (1961) 93-100
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gluco-corticoid hydrocortisone (compound F), the active adrenal steroid in the chick, produces enhanced hypercholesterolemia without affecting atherosclerosisU-13, 26, 27,47. This same effect was also demonstrated in the rabbit and most other animal species. The mineralo-corticoid desoxycorticosterone acetate (DCA) produces a slight hypertension in chicks and intensifies atherogenesis through this mechanism. When hypertension does not develop, no increase in severity of the vascular lesions is noted 11-13,26,41.
(3) Pancreas. Pancreatectomy is readily achieved in the chick, but pancreatectomized animals appear to behave to all intents and purposes like normal animals. T h e y do not show spontaneous hyperlipemia or hypercholesterolemia and they have normal blood-glucose levels and a normal glucose tolerance. However, on feeding cholesterol a subtle derangement of cholesterol metabolism can be shown. When cholesterol is given at a low percentage of the diet (0.25%) together with a low oil supplement, hypereholesterolemia and atherogenesis are similar in these pancreatectomized animals to those in the controls, but if a large (2%) cholesterol supplement and 5 % cottonseed oil are used, these pancreatectomized animals show enhanced hypercholesterolemia and atherosclerosis of the a o r t a and the coronary arteries 11,12,26,~7,42. As far as glucose metabolism is concerned, here too a subtle defect can be shown when these depancreatized animals are given c o m p o u n d F. Pancreatectomized animals respond to compound F with a three-fold increase in blood glucose as c o m p a r e d with normal hydrocortisone-treated animals. However, if hydrocortisone administration is combined with cholesterol feeding no e n h a n c e m e n t of atherogenesis can be producedn,13,42. These findings of a subtle disturbance of c a r b o h y d r a t e metabolism suggested an experimental analysis of the effects of insulin. I t was found t h a t insulin administered to normal non-diabetic animals on a cholesterol-oil supplemented, atherogenic diet does not influence atherogenesis. However, if insulin is given during the period of regression, after cholesterol feeding has been stopped, the regression of the atherosclerotic lesions is suppressed. This happens despite a reversal to normal of the dietinduced hypercholesterolemia. Preliminary experiments into the mechanisms of these insulin effects seem to indicate that hypoglycemia as such is involved in this phenomenon26, 59. However, more experiments with different hypoglycemia-producing agents will h a v e to be carried out to clarify this mechanism further. (d) Sex hormones. The hormones that show the most m a r k e d effect on experimental atherosclerosis in chicks are the estrogens20,25, 27. When cholesterol and estrogens are given concurrently, coronary atherogenesis is m a r k e d l y inhibited, despite the fact t h a t cholesterol-induced aorta atherogenesis proceeds unaffected4a, 5s. Moreover, it was also shown t h a t estrogen administration reverses coronary lesions previously induced b y cholesterol feeding, even when the cholesterol feeding is maintained during the period of estrogen exhibition44,49, 50. Here again, estrogen reversal of cholesterol-induced coronary atherosclerosis is associated with no such effect on aorta atherosclerotic lesions, provided the high-fat, high-cholesterol diet has a high protein content. J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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These experiments on estrogens and cholesterol-induced coronary atherosclerosis are of significance for several reasons. First, the plasma lipid d a t a recorded in these studies suggest t h a t the plasma ratio of cholesterol to phospholipids (C/P ratio) m a y be a significant factor in coronary atherogenesis. Thus, in all birds protected from coronary atherogenesis b y estrogen exhibition, plasma C/P ratios are at or near normal levels, in contrast to the control chicks fed cholesterol without estrogens, wherein C/P ratios are uniformly elevated. Further, the results of these experiments afford a lead to possible mechanisms in the well-known h u m a n sex-differential in susceptibility to coronary atherosclerosis 24. Finally, the results of these investigations highlight an i m p o r t a n t lesson for atherosclerosis research. So far, most investigators of experimental atherosclerosis had assumed t h a t the process of atherogenesis in different arterial beds is based upon essentially similar biological laws. Therefore, most workers confined their studies to the aorta and interpreted their findings there as applicable to atherogenesis in the coronary, renal, cerebral and other arteries. The results of these experiments on estrogens and coronary and aorta atherogenesis d e m o n s t r a t e beyond d o u b t t h a t this is a fundamental methodologic error. Since estrogen exhibition inhibits coronary atherogenesis in cholesterol-fed cockerels without exerting a n y effect on aorta atherogenesis, it m a y be concluded that different laws determine atherogenesis in the aorta and coronary arteries. Estrogens do not influence aortic atherosclerosis in chicks in which lesions were induced b y a high-cholesterol, high-fat, high-protein diet. However, if lesions were induced in animals b y a high-cholesterol, high-fat, low-protein diet and estrogens in large dosage are then exhibited, ulceration of aortic, particularly abdominal aortic, and coronary lesions were observedSX, 62. Thus, for the first time in experimental atherosclerosis, one other stage of atherosclerotic "complication" has been produced in the chicken. These experiments also indicated t h a t the ulceration of lesions in the animal m a y occur during the healing phase of atherosclerosis. This was shown b y carrying out serial weekly autopsies over a five week period. Preliminary d a t a indicate t h a t the tendency to ulceration is associated with definite morphologic differences between the appearance of the atherosclerotic lesion produced b y a high-protein diet and t h a t caused b y a low-protein diet. In the high-protein lesions a b u n d a n t fibrous connective tissue is laid down while the framework of the low-protein lesion is characterized b y a negligible fibroblastic activity. These data are being evaluated further. Estrogens continue to exert their protective action on coronary atherosclerosis in the chick even when combined with testosterone, adrenal hormones and pancreatectomy~5,45,4s, 55. The only regimen counteracting their effect is, as mentioned above, thiouracil-induced hypothyroidism26, 51. These findings seem to indicate t h a t a normal thyroid function is essential for the protective action of the estrogenic hormones on coronary atherosclerosis. CONCLUSION
This brief review of the main developments in experimental atherosclerosis in the chicken provides ample justification for using this animal species in experiments J. Atheroscler. Res., 1 (1961) 9 3 - 1 0 0
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attempting to analyze the pathogenesis and therapeutic value of different procedures and substances prior to their trial in man. It is, of course, clear beyond doubt that the last proof of the efficacy of any suggested regimen in man must ultimately rest on careful clinical trials. Animal experiments, in this regard, are merely a screening device. I t is also evident that the study of experimental atherosclerosis, and of the exogenous and endogenous factors influencing it, can yield knowledge of fundamental significance for the understanding of human atherosclerosis. Present knowledge does not permit us to state that the atherosclerosis problem has been solved by research along these lines. Much remains to be done. But it can be stated that considerable progress has been made in recent years since the epoch-making studies of ANITSCHKOW, and that this approach to the problem m a y ultimately bring the solution of the atherosclerosis problem, and with it the prevention, control and eventual elimination of this disease. ACKNOWLEDGEMENTS This research program of the department was made possible by generous support over the years by many private individuals and groups, and b y foundations and voluntary health agencies, as well as by grants from the Public Health Service of the United States Governement. The major sources of support included: The Michael Reese Research Foundation, the Lasker Foundation, The Chicago Heart Association, The American Heart Association and the National Heart Institute. Over the years a large group of physicians participated in the atherosclerosis research program. Among these, we wish to acknowledge particularly the most important contributions of Drs. D. V. DAUBER*, L. HORLICK, S. RODBARD AND J. STAMLER. These studies would not have been possible without the intelligent and skillful cooperation of the research assistants and technicians of the atherosclerosis research team. Especially deserving of mention are Mrs. C. BOLENE-WILLIAMS, Mrs. D. CENTURY AND Mr. P. JOHNSON, Research Assistants; and Miss C. KAKITA, Mrs. E. MILLER, Mrs. C. THOMPSON, Mrs. M. TRAVERS, Mrs. M. VANKINSCOTTAND Mr. G. CROWLEY,Technicians. SUMMARY The main morphologic features of spontaneous, cholesterol-induced and estrogeninduced atherosclerotic lesions in the chicken are described. The results of studies of dietary and hormonal influences on the formation and regression of these lesions are outlined. It is concluded that the results j astify the use of this animal species in the study of the pathogenesis of atherosclerosis and of the therapeutic value of different procedures and substances prior to their trial in man. * Deceased. J. Atheroscler. Res., 1 (1961) 93 100
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RESUM~
Les auteurs d6crivent les principales caract~ristiques morphologiques de ldsions ath6roscl6rotiques spontan6es provoqu6es chez le poussin par le cholest6rol et l'oestrog~ne. Ils exposent les r6sultats d'6tudes portant sur les influences di6t6tiques et hormonales sur la formation et la r6gression de ces 16sions. On conclut que les r6sultats justifient l'usage de cette espfice d'animaux pour 6tudier la pathogen&se de l'atMroscl6rose et la valeur thdrapeutique de diff6rentes m~thodes et substances avant de les 6prouver chez l'homme. ZUSAMMENFASSUNG
Die wichtigsten morphologischen Eigenttimlichkeiten der spontanen, bzw. durch Cholesterin oder Ostrogen hervorgerufenen krankhaften atherosklerotischen Ver/inderungen beim Kticken werden beschrieben. Die Ergebnisse der Untersuchungen tiber den Einfluss von Dffit und Hormonen auf die Entwicklung oder Rtickbildung dieser Ver~nderungen werden berichtet. Auf Grund dieser Ergebnisse erscheint das Kticken geeignet ffir das Studium der Pathogenese der Atherosklerose und des therapeutischen "vVertes yon verschiedenen Massnahmen und Substanzen vor ihrer Erprobung beim Menschen. REFERENCES E x c e p t for r e f e r e n c e to t h e r e v i e w b y ANITSCHKO~r, all o t h e r r e f e r e n c e s are confined to t h o s e p u b l i s h e d b y t h e d e p a r t m e n t . T h e w o r k of o t h e r s is referred to in t h e s e reports. T h e r e f e r e n c e s are divided into reviews, original contributions and preliminary notes, each arranged chronologically. 1 N. AmTSCHKOW, in E. V. C o w n R v , Arteriosclerosis, MacMillan, N e w York, t933, p. 271. Reviews 2 L . N . KATZ AND D. V. DAUBER, J. Mt. Sinai Hospital, 12 (1945) 382. 3 L . N . KATZ, J. SrAMLER AND L. HORLICK, Am. Practitioner, 1 (1950) 461. 4 L. N. KATZ, Trans. 5th Conference on Factors Regulating Blood Pressure, J o s i a h M a c y J r . F o u n d a t i o n , N e w Y o r k , 1951, p. 174. 5 L . N . KATZ, Circulation, 5 (t952) 101. 6 L. N. KATZ, J. STAMLER AND R. PICK, in Year Book Path. and Clin. Path., Chicago, 1951, p. 125 7 L. N. KATZ, J. STAMLER, R. PICK AND S. RODBARD, Lancet, 72 (1952) 326 ( P a r t I), 372 ( P a r t II). 8 L . N . KATZ, H. L. BLUMGART, Clinical Progress in Cardiovascular Disease, G r u n e a n d S t r a t t o n , N e w York, 1952, p. 9. 9 L, N. KATZ AND J. STAMLER, Experimental Atherosclerosis, C h a r l e s C. T h o m a s , Springfield, Ill., 1953. 10 L . N . KATZ, R . PICK, AND J. S'rAMLER, 2Plodern Concepts of Cardiovascular Disease, 23 (1954) 239. ll J. STAMLER, L. N. KATZ AND R. PICK, in H. SELYE AND G. HEUSER, dth Annual Report on Stress, A c t a Inc., M o n t r e a l , C a n a d a , 1954, p. 245. 1~ L . N . KATZ, J. STAMLER, aND R. PICK, Symposium on Atherosclerosis, N a t i o n a l A c a d e m y of Sciences, N a t i o n a l R e s e a r c h Council, W a s h i n g t o n , D.C., N . A . S . - N . R . C . P u b l i c a t i o n , 338 (1954) 236. x, j. STAMLER, L. N. I~ATZ, R . PICK AND S. RODBARD, Recent Progress in Hormone Research, Vol. 11, A c a d e m i c P r e s s Inc., N e w York, 1955, p. 4 0 t . 14 L . N . KATZ, A n n . Int. Med., 43 (1955) 930. 15 L. N. KATZ, in A. KEYS, Arteriosclerosis, a Symposium presented by the Minnesota Heart Association and the University of Minnesota, 1956, p. 25.
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L . N . KATZ, Circulation Research, 4 (1956) 123. L . N . KATZ, J. STAPLER AND R. PICK, J. A m . iVied. Assoc., 161 (1956) 536. L . N . KATZ, J. STAPLER AND R. PICK, Lancet, 76 (1956) 355. L . N . KATZ, J. STAPLER AND R. PICK, Federation Proc., 15 (1956) 885. J. STAPLER, R. PICK AND L. N. KATZ, A n n . N . Y . Acad. Sci., 64 (1956) 596. 21 L . N . KATZ, Symposium on Normal Limits and Functional Variations of the E CG. Proc. 3rd World Congress of Cardiology, Brussels, 1958, p. 403. 22 L . N . KATZ, J. STAPLER AND R. PICK, Festschrift Volume honoring $cademico Josef Charvat o n his 60th Birthday, S t a t e Medical Publishers, P r a g u e , 1958, p. 89. 23 L . N . KATZ, J. STAPLER AND R. PICK, Nutrition and Atherosclerosis, L e a a n d Febiger, P h i l a d e l p h i a , 1958. 24 J. STAPLER, R. PICK, L. N. KATZ, A. PICK AND B. M. KAPLAN, i n G. PINCUS, Hormones and Atherosclerosis, Academic Press, New York, 1959, C h a p t . 30, p. 423. 25 R. PICK, J. STAPLER AND L. 1kT. I~IATZ, in G. PINCUS, Hormones and A therosclerosis, A c a d e m i c Press, N e w York, 1959, Chapt. 18, p. 229. 26 J. STAPLER, R. PICK AND L. N. KATZ, in G. PINCUS, Hormones and Atherosclerosis, A c a d e m i c Press, N e w York, 1959, Chapt. 14, p. 173. 27 L . N . KATZ AND R. PICK, in H. T. BLUMENTHAL, The Hormonal Regulation of L i p i d Metabolism and its Etiological Significance, Charles C. T h o m a s , Springfield, II1., Chapt. 18, in t h e press. 16
17 18 19 20
Original Contributions 28 29
D . V . DAUBER AND L. N. KATZ, A..Vi.A. Arch. Pathol., 34 (1942) 937. D . V . DAUBER AND L. N. KATZ, A . M . A . Arch. Pathol., 36 (1943) 473.
30 31 32 33 34 35
38 39
D . V . DAUBER, A . 3 I . A . Arch. Pathol., 38 (1944) 46. L. HORLICK AND L. N. KATZ, J. Lab. Clin. Med., 33 (1948) 733. L. HORLICK, L. N. KATZ AND J. STAMLER, A m . Heart J., 37 (1949) 689. D . V . DAUBER, L. HORLICK AND L. N. KATZ, A m . Heart J., 38 (1949) 25. L. HORLICK AND L. N. KATZ, Am. Heart J., 38 (1949) 336. L. HORLICK AND L. N. KATZ, J. Lab. Clin. Med., 34 (1949) 1427. J. STAPLER, E. N. SILBER, A. J. MILLER, L. AKMAN, C. BOLENE AND L. N. KATZ, J. Lab. Clin. 2Vied., 35 (1950) 351. J. STAPLER, A. J. MILLER, L. AKMAN, E. N. SILBER, C. BOLENE AND L. N. KATE, Circulation, 2 (1950) 523. J. STAPLER AND L. N. KATZ, Circulation, 2 (1950) 705. S. RODBARD, L. ~'q-. KATZ, C. BOLENE, R. PICK, M. LOWENTHAL AND G. GROS, Circulation,
40 41 42 43 44 45 46
3 (1951) 867. S. RODBARD, C. BOLENE AND L. N. KATZ, Circulation, 4 (1951) 43. J. STAPLER, R. PICK AND L. N. KATZ, Circulation, 4 (1951) 262. J. STAPLER AND L. N. KATZ, Circulation, 4 (1951) 255. R. PICK, J. STAPLER, S. RODBARD AND L. ~N~.KATZ, Circulation, 6 (1952) 276. 1~. PICK, J. STAPLER, S. RODBARD AND L. N. KATZ, Circulation, 6 (1952) 858. J. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 1 (1953) 94. S. RODBARD, C. BOLENE-V~TILLIAMS, R. PICK AND L. 1~. KATZ, J. Lab. Clin. Med., 41 (1953)
s6
37
587. STAPLER, 1~. PICK AND L. N. KATZ, Circulation, 10 (1954) 237. STAPLER, R. PICK AND L. N. KATZ, Circulation, 10 (1954) 247. STAPLER, R. PICK AND L. N. KATZ, Circulation, 10 (1954) 251. PICK, J. STAMLER AND L. N. KATZ, Circulation Research, 5 (1957) 515. PICK, J. STAPLER AND L. N. KATZ, Circulation Research, 5 (1957) 510. STAMLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 442. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 447. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 6 (1958) 825. PICK, J. STAPLER, S. RODBARD AND L. N. KATZ, Circulation Research, 7 (1959) 202. STAPLER, R. PICK AND L. N. KATZ, Circulation Research, 7 (1959) 398. PICK, J. STAMLER AND L. N. KATZ, Circulation Research, 7 (1959) 866. HOJMAN, A. A. PELLEGRINO-IRALDI, M. R. MALINOW, l:{. PICK J. STAPLER AND L. N.
47 48 49 50 51 52 53 54 55 56 57 5s
J. J. J. R. R. J. J. J. R. J. R. D.
59
KATZ, A.,Vi.A. Arch. Pathol., 68 (1959) 533. J. STAMLER, R. PICK AND L. N. KATZ, Circulation Research, 8 (1960) 572. C. COHN, R. PICK AND L. N. KATZ, Circulation Research, i n t h e press.
60
Preliminary Notes 61 62 63
R. PICK, J. STAPLER AND L. N. KATZ, Federation Proc., 17 (1958) 124. R. PICK AND L. N. KATZ, Circulation, 18 (1958) 481. R. PICK AND L. N. KATZ, Federation Proc., 18 (1959) 120.
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