Naturally occurring atherosclerosis in howler monkeys (Alouatta caraya)

Journal of Atherosclerosis Research E l s e v i e r P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s

N A T U R A L L Y OCCURRING A T H E R O S C L E R O S I S IN H O W L E R MONKEYS (Alouatta caraya)

M. R. M A L I N O W AND C. A. M A R U F F O

Oregon Regional Primate Research Center, Beaverton, Ore. (U.S.A.) ( R e c e i v e d N o v e m b e r 4, 1965)

INTRODUCTION

There are few published observations on the incidence of atherosclerosis in freeranging subhuman primates. Studies have been reported on Papio 1, Cercocebus ~, and Cercopithecus 2. Our results in Alouatta 3 indicated that the occurrence of atherosclerosis was common in the aorta and great vessels (see below) of howler monkeys and that arterial lesions increased with body growth; males had a higher involvement than females, especially after puberty. Since the preliminary report was completed, a satisfactory age-grading of the animals was recognized and the possible effects of pregnancy studied. These findings are described here. MATERIAL AND METHODS

A field trip to procure and study specimens of howler monkeys (Alouatta caraya) was conducted in Northern Argentina. A laboratory where it was possible to perform the initial observations was organized at the Hydrobiology Station (Argentina) through the cooperation of the Argentine Department of Agriculture and Livestock. Three hundred fourteen free-ranging howler monkeys were studied. The animals were shot and brought to the laboratory within a few hours after death. Some hunters spared adult females with young; otherwise the sample was unbiased and representative of all ages and both sexes. A complete postmortem examination was performed on practically all animals. Arterial vessels were split longitudinally and rolled flat on Masslin towels. Whole organs and other representative tissue samples from each monkey were fixed in four percent formaldehyde and further studies were conducted in the United States. The aorta was examined when fresh and again after Sudan IV staining. Simultaneous observations were performed on the proximal portions of the branches emerging from the aortic arch and the initial common iliac arteries; all these aortic branches will be referred to together as "great vessels". The extent of sudanophilia and fibrotic lesions was determined b y comparison against graphs with a surface involvement previously determined by weighing. The lesions

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were graded from 0.5 + to 4 + according to color (sudanophilia) or thickness (fibrosis). Results were expressed as percent of surface involvement and also as an arbitrary score where the surface of sudanophilic and fibrotic lesions was multiplied by the respective grade in all arterial segments of each animal, and then added. With this method, "blind" readings on two different occasions of whole series of aortas agree within 10 % in 90 % of the cases. The heart was studied in 30 adult animals randomly selected from those with the highest aortic involvement. The coronary arteries were examined in two blocks of the myocardium, one above the A-V sulcus and one, immediately below. Two frozen sections of each were stained with Sudan IV-hematoxylin. The presence of arterial sudanophilia or fibrosis was interpreted as described 4. Sexual dimorphism has been observed in non-human primates 5-7, thus precluding the use of body mass as a reliable method to compare male and female monkeys of different weights, especially after puberty. Classification of the animals then was based on several criteria. Monkeys of each sex were first divided according to their weight. It was found that a sudden increase in testicular mass was experienced in animals above 4.0 kg of body weight and that no pregnancies were observed below this weight. Consequently, this mass was assumed to be indicative of puberty in howler monkeys. The presence of pregnancy was determined by visual inspection. Eight animals with no obvious pregnancy but with a large uterus have been set apart. They probably represent puerperal cases. One monkey had a very small uterus and has also been included in this miscellaneous group. Postpuberal male and female animals were classified according to wear of the first upper molar. This molar tooth has an early eruption time and is central to the grinding area of the dental arcade. The progressive wear changes as determined by visual inspection have been graded on a I - I V basis; this concords with another growth index derived from the dry weight of the lens (unpublished) (since only a few monkeys were observed in dental classes I I I and IV, they have been combined). Some animals have been used for anatomical or histological studies not related to the present paper. In another two monkeys, the aorta showed a dissecting aneurysm and an extensive medial calcification, respectively. These cases will be communicated s~parately. Two hundred ninety-two animals were found suitable for this investigation and are described here. The groups obtained are shown on Table I; the number of animals is lower than in Table I I because molar grading could not be performed in all. Results of the complete postmortem examination will be reported later. RESULTS

General findings Howler monkeys (Alouatta caraya) were abundant in the area. Subcutaneous fat was present in all animals and indicated a good nutritional state. As a whole they seemed healthy; gross pathology besides the vascular findings discussed below

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was u n c o m m o n . No i n d i c a t i o n of specific infections were observed. Males were the h e a v i e s t a n i m a l s weighing up to 10 kg; females a l w a y s weighed less t h a n 6.5 kg. The average weight of the b o d y a n d of o t h e r s t r u c t u r e s is shown in Table I.

A therosclerotic lesions in the aorta and great vessels I n a few a n i m a l s atherosclerosis was d e t e c t e d b y n a k e d eye inspection. The n u m b e r of lesions, though, was more a p p a r e n t after S u d a n staining. I n t i m a l sudanophilia covered up to 10 % of the surface of the s t u d i e d vessels. These lesions corr e s p o n d e d m i c r o s c o p i c a l l y to regions w i t h f a t - l o a d e d cells b e n e a t h the e n d o t h e l i u m w i t h o u t fibrosis or elastic changes. Circumscribed raised plaques r a n g e d from 0.5 to 5 % of the o b s e r v e d a r t e r i a l surface a n d were generally l o c a t e d a t the ostia of the b r a n c h e s emerging from t h e aortic arch; m o s t of these plaques e x h i b i t e d different grades of sudanophilia. Several changes were o b s e r v e d microscopically: in the early types, a small s u b i n t i m a l deposit of lipids w i t h slight fibrosis was present a n d no modification in the elastic lamellae or in the m e d i a was found. I n the m o s t a d v a n c e d plaques, large d e p o s i t s of lipids were covered b y a highly fibrotic s u b i n t i m a l cap; the lipids e x t e n d e d also into the m e d i a a n d the i n t e r n a l elastic lamellae were disr u p t e d or d i s a p p e a r e d in spots. I n t i m a l changes were v e r y c o m m o n in b o t h sexes, especially a b o v e 4.0 k g b o d y weight. The p e r c e n t incidence of lesions as o b s e r v e d in all a n i m a l s is shown on Table I I . T h e e a r l i e s t changes were found in t h e thoracic aorta; t h e y were n e x t present in t h e

Fig. 1. Aortic arch of an adult male howler monkey showing atherosclerosis. Fibrotic strands are clearly observed in the plaque. The vessel has been opened flat and the ascending aorta is on the left; one of the branches of the arch is seen above. S, sudanophilia: F, fibrosis. Scale in cm.

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Fig. 2. Diffuse atherosclerotic involvement of the thoracic aorta in an adult m~le howler monkey. Same view and symbols as in Fig. 1. branches from the aortic arch and in the abdominal aorta. Involvement of the common iliac arteries was generally similar to the abdominal aorta. Extension of the process also followed a like order. Fig. 7 shows the surface involved in male and in non-pregnant female monkeys. Although strict comparisons between males and females cannot be based on weight because of sexual dimorphism, intimal sudanophilia seemed to occur earlier and more extensively in males; no abdominal lesions were observed in females. In order to compare lesions in both sexes, relative age has to be considered. Table I I I shows that no differences were observed in prepuberal animals. Postpuberally, though, males had a higher involvement than females, mainly because of more extensive sudanophilia. The differences, although still present, were not significant in the oldest category due to the large variances.

Pregnant and non-pregnant monkeys Pregnant animals of postpuberal class 3 had a lower involvement of the aorta and the great vessels than corresponding non-pregnant monkeys (Table IV). When successive classes are considered, though, the sudanophilic lesions progressed in the non-pregnant animals while no increase with age was observed in the pregnant

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Fig. 3. Large atherosclerotic plaque situated b e t w e e n the arch and the descending a o r t a of an adult male howler monkey. This localized lesion was the m o s t p r o m i n e n t one observed in the p r e s e n t series. Symbols as in Fig. 1.

m o n k e y s . R e s u l t s in the " m i s c e l l a n e o u s " group (see methods) are b a s e d on too few a n i m a l s to be of significance, b u t " s c o r e s " are i n t e r m e d i a t e b e t w e e n p r e g n a n t a n d n o n - p r e g n a n t animals.

Fig. 4. Aortic arch of an adult male howler m o n k e y showing diffuse fibrosis. Small areas of s u d a n o philia are present. Same view and s y m b o l s as in Fig. 1.

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NATURALLYOCCURRINGATHEROSCLEROSISIN HOWLER MONKEYS

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Fig. 5. Frozen section of a sudanophilic plaque in the aorta of an adult male howler monkey. Lipid material is observed mainly in the media; no intimal thickening is present. Sudan IV-hematoxylin; x 120.

Coronary atherosclerosis The coronary arteries were studied in 30 hearts (15 of each sex) from adult monkeys. In approximately one-half of these, a few arteries showed minimal fibrosis (proliferated "spindle cells") which were difficult to separate from the "normal ageing process". In two instances, a very early sudanophilic deposit was observed in the intima. The distribution of the changes was the same in both sexes and was unrelated to dental wear. DISCUSSION Howler monkeys are the largest nonhuman primates of the New World. Taxonomically, they belong to the family of the Cebidae, subfamily Alouattinae which has only one genus (Alouatta) with several species. These animals are found in tropical forests extending from the southern regions of Mexico to Northern Argentina s. They are arboreal, essentially vegetarian monkeys with prehensile tail and although they reportedly dwell poorly under laboratory conditions, we have presently been able to maintain two Alouatta caraya in apparent good health for over one year at this institution (unpublished). An extensive bibliography has accumulated on Alouattinae but few pathological studies exclusive of tropical diseases have been performed and we are not aware that the arterial system has been observed 3. Age of free-ranging primates can only be estimated. Several points seem well established, though, in our material. Sexual maturation probably occurred around 4.0 kg of body weight as attested by the increase in testicular mass or by the occurrence of pregnancy. Wear of the permanent dentition is useful for assessing age 9. Independent studies performed on the lens of the present monkeys (unpublished) showed that a straight fit was present between wear of the first permanent upper molar and the dry weight of the lens, a tissue which increases its mass with age 10.

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Fig. 6. Fibrotic plaque in the aorta of all adult male howler monkey. The intima is thickened; spindle cells are seen beneath an apparently normal endothelium. Clear vacuoles probably representing lipids, as well as increased rmmber of fibres can be detected irl the subintima. The elastic internal lamella is disrupted and proliferation of connective cells can be observed in the inner media. Gomori's trichrome; x 150. B o d y mass differed greatly in the monkeys; males attaining weights up to 10 kg while females did not exceed 6.5 kg. This sexual dimorphism has been observed in other primatesS,6, 7. Nevertheless, b y combining information from b o d y weight, sexual development, dental wear, and d r y weight of the lens, pre- and postpuberal animals could be classified into comparable groups of presumably similar age. Although atherosclerosis has been observed in over a dozen genus of s u b h u m a n primates 11, there are only two reports on the prevalence of the disease in large series of free-ranging animals. MCGILL et aL 1 found a high incidence of aortic lesions in 86 male and 74 female postnatal baboons (Papio anubis or Papio doguera). These authors could not observe a n y consistent trend with age nor a n y relation to sex or gestation. STRONG AND TAPPEN 2 found aortic lesions in 30 out of 49 Cercopithecus albigena and in 5 of 64 Cercopithecus ascanius; in the former, the incidence increased with g r o w t h of the animals, b u t no sex-related differences were observed. The findings reported here are somewhat at variance, probably because of our larger sample. For instance, MCGILL et al. observed 41 male and 26 female adult baboons while the present material represents 131 and 99 sexually mature males and females, respectively. Alternatively, a species difference could also account for the discrepancy. Results in howler m o n k e y s show t h a t atherosclerotic "scoring" of the aorta and the great vessels was progressively higher with "age", especially in relation with the sudanophilic lesions. Although the involvement was more i m p o r t a n t in males t h a n in females, the differences were not statistically significant in the oldest animals. Such sexual disparity in the involvement of the aorta has also been observed in the human, although it is not constant in all races and tends to disappear with agel2,1~, 14. I n other species, n o t a b l y the chicken, there are clear sex-related aortic lesionsl5,16.

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The influence of pregnancy is difficult to interpret and further studies are necessary. Although few animals were available, the impression was that monkeys which were pregnant when observed had less atheroselerosis than those females which were not pregnant at the time of death. Microscopic studies performed in the uterus and the mammary glands of these non-pregnant monkeys showed evidences of past gestations (disrupted muscle cells and fibrotic scars in the cervix, and hyalinosis and hypertrophy of the uterine arteries; a few of these animals showed lactating mammary j . Atheroscler. Res., 6 (1966) 368-380

NATURALLY OCCURRING ATHEROSCLEROSIS IN HOWLER MONKEYS

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glands). Consequently, the apparent reduction of arterial sudanophilia suggests a cyclic effect of estrogenic, corticoid or other hormones which are increased during pregnancy 17. In this connection, estrogens and adrenal steroids have been able to decrease the incidence of experimental atherosclerosis in several species is. As a related observation, it was interesting to note that when groups of postpuberal males were compared with those of non-pregnant females, results were different than when comparisons were made against all females. Although the average of intimal lesions were still higher in the former, statistical significance was lost, partly, at least, because of the large variances and also partly from the deletion of the pregnant monkeys. Coronary lesions were minimal in the animals studied. The observation suggests that the disease is relatively independent in both territories, as already reported in other species including man 19. The fact that no differences related to age or sex could be documented is compatible with the early characteristic of the changes but m a y also represent a feature of the coronary arteries in Alouatta. As indicated above, howler monkeys are considered to be mainly vegetarians and no animal material has been observed in their stomach contents s. The occurrence of atherosclerosis in the absence of exogenous cholesterol has already been reported in other speciesl6, 20. Because of the high incidence of gross intimal changes, the anatomical similarities with early human aortic lesions, the possible sex-related pathology, the lack of apparent infectious and parasitic diseases, and the possibility of laboratory observations, Alouatta caraya seems to be a suitable primate for experimental studies on atherosclerosis. ACKNOWLEDGEMENTS

We are grateful to Dr. I. T. Marini, Director of the Department of Fish and Wildlife Conservation, Argentina, who kindly granted us the use of the laboratory and to Dr. A. L. Rosensvaig, Director of the Hydrobiology Station, who provided continuous support and help during the various phases of this study. We are also thankful to Mr. Alberto Segal who assisted us in Buenos Aires, Argentina, with several aspects of this project as well as to Mr. Richard Gearhart from the John C. Higgins Foundation, Portland, Oregon, for his kind interest in the field work. Dr. L. Lusted, Chairman and Mr. Dennis Lindgren, Division of Biomathematics were instrumental in computation of the data. Publication number 124 of the Oregon Regional Primate Research Center; aided by the Operating Grant No. FR00163 of the U. S. Public Health Service. SUMMARY

The aorta, the proximal portions of the branches from the aortic arch as well as the initial common iliac arteries have been studied in 292 howler monkeys (Alouatta caraya), essentially a vegetarian animal. Atherosclerotic lesions were commonly found. J. Atheroscler. Res., 6 (1966) 368-380

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M. R. MALINOW, C. A. MARUFFO

The incidence increased progressively with age and was higher in males. An apparent decrease was present in animals which were pregnant

a t t h e t i m e of o b s e r v a t i o n .

O n l y m i n i m a l l e s i o n s w e r e d e t e c t e d i n t h e c o r o n a r y a r t e r i e s of 30 a n i m a l s r a n d o m l y selected among adult monkeys showing the highest aortic involvement. No differences related to sex or age were observed in the coronary arteries. REFERENCES 1 H. C. MCGILL, JR., J. P. STRONG, R. L. HOLMAN AND N. T. WERTHESSEN, Circulation Res., 8 (1960) 670. 2 j . p. STRONG AND N. C. TAPPEN, Arch. Pathol., 79 (1965) 199. 3 M. R. MALINOW AND C. A. MARUFFO, Nature, 206 (1965) 948. 4 M. R. MALINOW, J. A. MOGUILEVSKY, B. LEMA, G. E. BUR AND A. ERENFRYD, Circulation Res., 9 (1961) 204. 5 j . A. GAVAN, Human Biol., 25 (1953) 93. s G. VAN WAGENEN AND H. R. CATCHeOLE, Am. J. Phys. Anthropol., 14 (1956) 245. 7 A. H. SCHULTZ, in A. HOFER, A. H. SCHULTZ AND D. STARCK (EDs.), Primatologia, S. Karger, Basel, New York, 1956, p. 887. 8 W. C. O. HILL, Primates, Comparative Anatomy and Taxonomy, Vol. 5 (Cebidae), Part B, Univ. Press, Edinburgh, 1962. 9 A. E. W. MILES, in: D. R. BROTHWELL (ED.), Symposia of the Study of Human Biology, Macmillan, New York, 5 (1963) 191. 10 R. D. LORD, JR., J. Wildlife Management, 23 (1959) 358. 11 M. R. MALINOW, Folia Primatol., 3 (1965) 277. 12 j . p. STRONG, J. WAINWRIGHT AND H. C. MC GILL, JR., Exptl. Mol. Pathol., Suppl. 1 (1963) 15. 13 C. RESTREPO, J. P. STRONG AND H. C. McGILL, JR., J. Atheroscler. Res., 4 (1964) 213. 14 j . p. STRONG, J. WAINWRIGHT AND H. C. MCGILL, JR., Circulation, 20 (1959) 1118. 15 DAUBER, A. M. A. Arch. Pathol., 38 (1944) 46. 16 M. R. MALINOW, B. LEMA AND S. J. KAPLAN, g. Atheroscler. Res., 3 (1963) 321. i7 M. X. ZARROW, in: W. C. YOUNG (Ed.), Sex and Internal Secretions, Williams and Wilkins, Baltimore, 3rd edition, Vol. 2 (1961) 958. 18 M. R. MALINOW, Advan. Pharmacol., 2 (1963) 211. 19 I. GORE, W. B. ~ROBERTSON, A. E. HIRST, G. G. HADLEY AND Y. KOSEKI, Am. J. Pathol., 36 (1960) 559. 2o W. W. HARTROFT AND W. A. THOMAS in: M. SANDLER (ED.), Atherosclerosis and its Origin, Academic Press, New York, 1963, p. 439.

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