the natural history of atherosclerosis

The Natural History of AtherosclerosisBy MARCEL M. VASTESAEGER, M.D., AND R. DELCOURT, M.D.

A LTHOUGH unceasing attempts have beenj L made to increase the knowledge of hu-man and experimental atherosclerosis, thepathogenesis of this disease is still far frombeing thoroughly understood. The uncertaintyof present conceptions concerning atherogene-sis is emphasized by the rapid successionduring the last 20 years of several theoriesallotting a predominant role to different sub-stances such as cholesterol, animal fats, sat-urated fatty acids, and nonessential fattyacids, and the series has probably not yetcome to an end.

Hitherto, epidemiologic investigations havebeen quite anthropocentric: although the inci-dence of clinical atheroselerosis among var-ious human populations of the world is rela-tively well known, the frequency if itsspontaneous occurrence in the entire group ofanimals having a specialized circulatory sys-tem is almost completely unknown. In otherwords, most of the natural history of athero-sclerosis is still to be discovered. The purposeof this paper is to sum up the fragmentaryknowledge in this field, and to pose someproblems the solution of which might be ofinterest for the etiology of atherosclerosis.

In order to avoid confusion, it may be op-portune to define once more arteriosclerosisand atherosclerosis. As, proposed by Lobstein'arteriosclerosis is a generic term, designatingseveral arterial degenerative processes givingrise not only to a thickening, but also to aloss of elasticity of the vessel wall (diffuseintimal sclerosis, Mdnckeberg 's2 medial scle-rosis, focal atheroselerosis, etc.). Unfortu-nately, too many workers have discarded thegeneric sense of arteriosclerosis and used this

From the Centre d 'hude des Maladies des ArteresCoronaires, Department of Cardiology and Depart-ment of Clinical Pathology, Hopital d 'Ixelles,Brussels, Belgium.

Circulation, Volume XXVI, November 1962

term when alluding exclusively to one or an-other of the various lesions of this condition.Such habits have made literature more andmore confusing.As a matter of fact, the only valuable way

of defining each kind of arterial degenerativelesion is based on histologic criteria. This iswhy, referring to etymology, and accordingto the recent nomenclature of the WorldHealth Organization,3 we shall confine our-selves in this paper to the following defini-tions:

1. Intimal sclerosis: diffuse fibrous thicken-ing of the intima, generally associated withdegeneration of the intimal elastic membraneand with sclerosis of the mnedia. Grossly, dif-fuse intimal sclerosis gives rise to some dila-tation and to lengthening (unrolling) of theinvolved vessels, but the lumen is not im-paired.

2. Atheromna: strictly focal lesion (e'tVa' =tumor ), characterized by the presence oflipids (a'Thj'py = mush), conferring to theplaque a grossly yellowish color.

3. Atherosclerosis: more recent word intro-duced by Marchand4 and referring to a lesionintermediate between intimal selerosis andatheroma. Usua.lly focal, atherosclerosis hasan important fibrous component and con-stantly shows lipid deposits. In their ad-vanced stages, atheroma and atherosclerosisgrossly impair the lumen of the vessel.As a rule, atherosclerosis is less localized

in animals than in man. Especially in birds,stenosing atheroselerosis is much more diffusethan usually observed in human arteries(fig. 1).The diffuseness of spontaneous atheroscle-

rosis in animals often makes it very difficultto establish a clear-cut difference betweenmere intimal sclerosis and the first stages ofatherosclerosis. This explains why it is very

841

V4 YASTE,SALENE,1)ELCU(R)IlZ

Figure 1Coroner! artery of (otljeeon tee duck (Dendrocygnus javaniicus Ahor.]).A. Coneentrie thick-mug of the itirln : the hinun1(9 1 stiocytes wuithiriletr cytoplasm sh/io the atheroselerotie natun'C

ofthi's lesion. Gou X>2t7. B. Another section. in

the same erterilq shoo-ingfJ importance tidt (liffntst-

ness of the lipid deposits. Stdorla bleeak B X27

difficult, if niot im-possitAble, to dissociate thenatur al history of atherosclerosis from thenataural history of dliffuise intiinial selerosis.The Avor k carried out for niearlyA half a

cen.jturv at the Zoo of Philadelphia has builtmtiost of our presentklnowledge of spontaneouLsatheroselerosis in wil(d aniimals. Fox,5'6 was

thIeI filst to shio 1ht.veI.Xot iaInIIIa1IIIils a 11(Iibirds livilig i i cat it v i iit iv ac1il Id t,(legelereati_V(e arte-rial li1siols. (ouit --11111g'. th10wock of' Fox, Baltt(1iffe ( 0rmihi) showedthat tle ineideliee (,f (legCel,ieatvx' aterialphleC tllona seevs; toio e(aS( lrO-flnrt jioallxt.vxv.itl tile rate of' over 1l)opllation of zoos, and(iAw-itlh the str'esses t-o wheli tI-le animials arieSu1)4jet(ed. Eeei itlY, Ealct -lifit'c'-,Yerasi II idc1(7ani-d lE'lliot t' iinsisted upIol I lie, icnireasi ig Ie-cuei(eyll(v of steioEsiIg eorounrx lesimns Hi tlieIhomel(othernic anIimal's of the PhiladelhliaZV0O since 1950, inlvoking agai(n aIs Ietl Cause

[avorllou the vxol iti loll of these elsioiis I lie'V lug(Ieist o tne ejot ivet(,t]phvanimal] plop-

lat.ioll.

In1- the oi-ller hanilld , spoiiau leons Itt inalselerosis or athlcrosrlIteosis lhas beent. dese Insin iny species of domnestie ail;fmoals, 1lir(ll aildni.iiiiiials, alike i(ihiekeli ill)it,1 d{log,11' 12and pigeoli1.1" So,on.einax oiisiilde it fils1111vestablished thfiat mI(ost sp)cies o fiomeotl)hrlni'animals that: ni has doinest:hafite.d orl reducedto Car)tivity, Jimov s)oiltxtleousl.x (lexeh)l) 50m111towin of arCCi 1)8(1serss .\Vith]54,ia

fhil)-potauittl).oiiits ieeessary forL the coiupllet'e11iderstaialidl1 (of thle re.nesis of altelial de

(rei('Lerativte eeesses ae s mid of eni ntIatulllistolry TeMaiJi Ob'senrilet.

tuinde:r thie mIpi(s s of tilew il,iegiaj (celerfror theC Studly ()f (`oronary- Artery I)iseasc i

svstemiatic researelh jilto) the arter ial degeeIra.el-tire ma-ifestat iolXs ii the anlinial. killn-domhans b:ceen uniIdertakecn for a fe xvv eari-s. I I viewv

*Tlese stuSdies couu(II C8( 1T Icir idout only bec.lilsoof the Very obliging hell) of 1m1aty11y oigaiizit-.tioalls ilndifferenit coiiitries. Aee wish to thank sotouig otlcrs:The Intrn1atelt;iolall Soce;Wtv of (ald-iolog) (I)r-s. Whiteno]l Kat.tz); Tfe Aiitwerp) Zoo (Mr. V.an den BerglaInld Pi-of. Morhimatl-ins); Thie ''lJustiituluto LatinioAincrica:o de \Arttomia patologi,jc II of Me'eico (PIrofCostero); The Laboratory of Paithology of thelrnw;sels 'University (Prof. Distiai aniid Dr. Pavinenitier); The med(ical Laboratory of Stanfle) v lle (CoII-go); Thie capturing camp of Einpulti (Conlgo), Mr.d(o AMediiia; The Commilission of Coopa-trative Alnltomy(Kaiisas, USA), Dr. A[aizl<e; Tue Scienitific Peseaireblilnstitulte of Pilill,itz (Fiancec), MAr. Bari6ty andAMr. PIcrcier; The MArlrine Biology Stationl of l?oscotfi(France) ; Thle ArAsterdani Zoo Toil Veteriina.ry Schoolof Utrieclit s Uniiversit3, Prof. Zw art.

Circulation, Volumc XX V., Novmnber 1.962

1; 42)

NATURAL HISTORY OF ATHEROSCLEROSIS

of the huge field to be explored, these inves-tig-ations are still in their first stages, and thespecimens hitherto examnined of invertebratesare too few to fuirnish valuable data. Amongvertebrates, however, a few points worthv ofiniterest alreadv have been recorded.

Material and MethodsThe gathered imaterial consists first of the hearts

aind coronarv arteries of 403 captive wild verte-brates, that died in the Zoo of Antwerp or at thecapturing camiip of Epulu, and the aortas of 72 ofthem (table 1). The anatomy as well as the pateneyof the coronary arterial tree of most of these ani-toals was investig,ated by postmiortem arteriog-raphy.A second group of imaterial includes the heart

anid coronary arteries of 144 vertebrates living infreedom and killed in their natural habitat. Theaortas of 62 of them -were also examined (table 2).

TIn both groups of animilals, the proximal seg-ments of the main coronary trunks were removedand fixed in formalin. Paraffin sections were pre-pared and stained with Masson's triehronme stain(iron bematoxylin-ponceau-light green), and withWeigert's resorein-fuehsin for the staining ofelastic tissue. Very often, Gabe's method (aldehyde-fuchsin) was used for the simultaneous stainingof elastic and connective tissue. We occasionallvprepared frozen sections in order to stain lipidswith Sudan black B.The partially dehydrated aortas were stained in

a saturated solution of Sudan IV in 70 per centethyl alcohol for 10 minutes, and then differen-tiated in 70 per cent ethyl alcohol for 24 hours,in order to remove excess stain. Frozen sectionswere made in the stained areas.

Thirdly, in 88 of the captive anim-lals and in 51of the free ones, blood samples were obtained byvenous or by cardiac puncture, and the followingdeterminations were made: lipidograim, total bloodcholesterol, cholesterol/phospholipid ratio, choles-terol fraction bound to the beta-lipoproteins, and,in many cases, activity of the clearing factor, asdetermnined by Hahn's technic.l4 Mfethods were de-scribed in a previous paper.15

ResultsDegenerative Arterial Processes in PoikilothermicAnimals

Elastosis of the initernal elastic membraneof the coronary arteries was observed in some

sharks (Isurus Nasus Donnaterre). In the in-volved arteries, elastosis was an isolated proc-ess, withlout any intimal sclerosis.Among bony fishes, however, much more


Table 1Captire Vertebrates

1. Reptiles: 38 specimens, ineludiiig represenitatives ofCheloniidae (10)Crocodylidae (4)Varanidae (3)Lacertidae (4)Iguanidae (1)Serpenits (16)

2. Birds: 123 specimeins, includinig represenitatives ofStuthionifornies (2)Galliformes (15)Lariformes (3)Balearicidae (8)Sphenisciformes (2)Anseriformes (27)Cicoiiidae (30)Falconidae (6)Coraciiformes (20)Passeriformes (10)

3. Mammals: 242 specimens, iincidiigrepresentatives of

Marsupialia (3)Carnivores (28)Pholidota (6)Pininipedia (8)Perissodactyla (8)Suidae (6)Ruminants (75)Rodents (10)Proboscidae (3)Primates (95)

Table 2Vertebrates in Freedom

1. Fishes: 40 specimens, iineludinig represenitatives ofSelachae (1)Anguillidae (3)Cyprinidae (20)Thunnidae (10)Istiophorus (6)

2. Birds: 36 specimens, ineludinigSphenisciformes (29)Ciconiidae (2)Coraciiformes (5)

representatives of

3. AMaminals: 68 specimens, includinigrepresentatives of

Carnivores (3)Suidae (35)Ruminants (9)Primates (21)

characterized lesions were observed. This wasespecially the case in the coronary arteries ofsomne fishes (bluefin tuns: Thunnus ThynniusL.). The first changes eonsisted in circularor semiicircular nodules of hyperplastic inti-miial tissue projecting into the vessel s luien

843

VA4VrAESAEGER, )ElCOfIT()UTRT

Figure 2AFat l intin4a/l 'cItctoau itt; 1/te rujtt c4't44t4r4 otertt1-av( /' t it1if)4 1 ( /i sI . I Iltup 'tXc .I X e-j, {/ I ttl}l ;7. leifA

t1igure znFJ"ca/ i14ti4441/l st'l'tr'osis i?/ tltt r'igh/4t c'r)4't0()ry', arte'-ios °!/ (4 1tioin/ fish. llt itJct-t X t).

Figure 2CAbundi anlt lipid deposits in the thi kerned mailcoronary t? n/k of a immature Thnn?ns. Sudanb1)/c/1 B X 2).5.

(fig. 2A ) Tllw internal elatst i' mIlcbllltl)'al)mi(lIerlleathi the inltial proliei-ations wa,sso0li44mes rllptulle( aiid its frarinent s weredlisJ)erse(l ill the hvyLerlplastie, intinia. Al-tflhou(g. fibroblasts ta-nd elolagyenois suhstaiieewxxeve the mi-laiin constituents of tle iollvlules, thepeseis(iiee of iinuerous histioeytes witlh tlear-oxytopla4S1in -very often eonferre( to tihle lesiou-S

(lefillite tendencvy towatid atherosclerosis(fi'. 213). Trplse (ealnge0s, elosel) reSelhl)liillgthliose reee)itly (dmlesribed inI spawvning' PacifficSablions bV Robertso. Wexlexr, alill Miller,'4WClre0 found ill till of the' 10 illVtstlgated tlnl-nielS rPlwey were xoun c, intiluiilt lure speleliliellsabmit :3 feet lo(,n, eatught in thle l)b-v of B'iscaIV(Fainee) The main eoronary rtlerv of two

of thiem shiowe(d wilespreadl atherosclerosiswitlh abundant 4xtrial'T4ll1flar ljpid. d1e'posits(fig. 2C).

(Certai llial)iallianu reptiles, iII (raptix'ity atletlst, eal also (develop dlifflse ilntilall selero-sis. This is tlhe (ease x ith eeritaiu opliidiainsof the typ)llo)s giolup: tlle coronary arteriesof a, largte ainaeoida (Eutoec.s marinaus L.)that died ifth ArlipA ver Zoo, slowevd fra"-mneltation of thie intern'al. elastic( 111(lineb)rancxvitlh slight fibrous, tlhickeninlg of the iltimna.

Focal initiimial thickenlieng witlh ruiptured in-terutal elastic inenibrane was also fotunid in a

main coronary trunk of a 9-foot crocodile(Cr'oco(dilats iloticts) living for mtiairany mnonithis

Circulation, Volum e X\X VI, Nov mbe r 19432

8H44

NATURAL HISTORY 1F ArlAHEROSCLEROSIS

in captivity at the Epulu camiip (Congcro). Inith1e lesiolns, abmndant listiocytes witli elearcytoplasmn also sugg(-est a defilite ten(ledilrtowarl atherosclerosis (fi(g. 3A aiid B). 1-iithis animiiial, gross sills of coroiary s-clerosisws,ere see-n.

For fishes as well as for reptiles, the nmimh)er of investigated specimelnes is too snall fotrainestimnationi of thle freqneiiev of arterilosle-ro(sis inl thlese (grolupsll of -verltebrylates.

Frequency of Atherosclerosis among Warm-Blooded Vertebrates

,ortie Atheoscletieosis

Siidanophilic fatty streaks of atleroscl-rot' nature are relativelv ommll11on0 ill thIeaorta of wvild mraImlInals ail(1 birds, and werepreseInt in nerl1y 30 per cent of tfle exallledsIvecinleiis (table 3).

Aimioi cai)tive aniiiimals, the lipid (Iel)Ositswere very strik6ing ii thle aorta of a rlhea(Rhcau IRotschild Brab.), of a l)eliean (1P%i-cU/ins Speick), of two wild boar (Su?s ScrafuL.), of a 30-year-old eanmel (Ca}me/tus bactri-anus LI.), of ani ai1dult biiffalo (Bubalus b)ubalisL.) of a yak (Roephagrus grunWiens L.), of aii

oldI baboon (Pupio Paplo Desmarest), and(1 ofa youmig mnale ebiinpanzee (Pan Sch/iuvcfurtl/hii [Cigi])Of the wild birds shot in freedom, nlolne(

shiowedl any sio'n of aortic atherosclertosis.Verv slihlt lipid (leposits wvere observed i1nthle aorta of eight of 35 wild boar (Sus Scnaofui

4 ortic .4 therosclerosis

Bf

Figure 3A. and 13. Focal intiwald sc lcrit in the caranarqairteries of at. 6-foot cap/iet ecrocodul e. Thme 1??nt r-0/Ifs hitioctqtcs With a ct atl? cyjotplasmnsShow cc dcj1Ji-

il t e/t anned /h eWheo lerrosis. WolViqed A,X 2.5; B, X 100.

i.) sihot ill theilr nIaltnlll'- habitat. Of 1three'(leer, ani adult feimale showed moreICIispici-aius faitty- streaks, (dissemimlite(l ill its thoracicaild abdomfinal aorta.

Table 3

1. Reptiles

2. Birds

3. NlanimialsMarsupi.aliaCanilivoresPerissodactyla.SuidaeRumninriantsRodentsPrimates

Total

Captive specimensNormal Atherosclerotic

3

4 4

1 -14

14 618 13

48 24

Free specimensNormal Atherosclerotic

0

28 77 1

10

47 15

Cireulation, Volume AX VI, Nouvnilber 1962

Total

9

162

3628

38

13*4

8 0

846VATSESAE%JER,. DELCOJR T

Table 4(o.obus MonAeks

No. Genus

I49

4)

8

C. BadiusC. Ba diusC. BadiusC. BadiusC. AngolensisC. AngolensisC. AnigolensisC. Aiigoleiisis

Table 5Inatimeal Sclerosis in Primates

AorticSex atherosclerosis

FF

F +A:tFMl

++-

Amoong free primates, in lnonie of the fi-velIemnuridae (one Perodicticus Potto [Gmiielland four Galago Demidovi [Fischer]) was

ani sudaniophilic spot to be fo-und, in spiteof the fact that the serumii of one of the gala-gYos was turbid. Of the 11 African monhkeys.two adult females of Colobus (Colobus auigo-

lensis), a youngomale of Aseanius (Cercopithl-cts ascanius [Audebert]), and an old female

of Mangabey (Cercocebus aterrim us [Oude-mnans]) showed widespread fatty streaks inthe thoracic aorta and less important ones inthe abdominal aorta (table 4).Of the five examined ehinmpanzees shot in

freedom, two had localized atheroscleroticlesions in the thoracie aorta: the lesions were

larger anid more important in a young adultfemale than in a somewhat older adult muale.

Neither in eaptive nor in free aniiials were

aortie lesions complicated by caleification, byimitramural hemorrhage, or by thronmbosis.The aoirtie arch was rarelv affected alone, anidit was rather commonier to find fatty patchesdisseiminated along the deseending portion ofthe thoracic aorta and stretching along theabdominal portion. Such a dispersion of thelesions was observed in birds as well as inmaiymals; in ungulata. at least, the thoracicaorta seemed to be affected sooner thain theabdomuinal portion.

Coronary Atherosclerosis

Coronary atheroselerosis is rare in our ma-

terial; it was found in onlv nine of the 484Nvarin-blooded vertebrates, i.e.. about 2 per

oeut of the examinedi speeimeiis. All of theaiffected indiviiduals were captive: two were

LemuridaeCallitrichidae aind CebidaeCereopithecidnePongidae

Withintimalsclerosis

3233965

Total

8923_515401

125

vegetarian mammin-als (a mouffion, Ammotragus.Lervia [Pallas], and a tapir, Tapirus InlldictusDesmarest); three were omnivorous mammrnals(two chimnpanizees, Pani Sch weitfurthii [Gigl],anid one little panda, Ailurus fudgen1s [Cuv.])the other three were vegetarian birds (acrownied crane, Balearica pavoninta (LA), andthree tree ducks, Dend-rocygn?a javanica[Horf.].Atherosclerosis of the nmouffloi 's coroniary

tree was very slight anid consisted exclusivelyof snall sudanophilic spots in the in-tima sdiffuse thickeninig. More widespread in theaniterior deseending braneh and in the eircumi-flex braneh of the left coronary artery, werethe lesions of the tapir: they eonsisted notonily of lipid deposits in the thickened fibrousintimna but also of raised areas forminig realatheromatous plaques (fig. 4).The muore conspicuous lesions of the

chimupanzees were hunman-like atheromatousplaques with foam cells and cholesterol crys-tals. In the little panida, however, they werequite differenit: despite their diffuseness! andthe absence of atheroma, an oeclusive thromn-bosis occurred in the anterior descendingbranieh and killed the animal.17 In this case,also, foam eells and cholesterol crvstals werepresent in the thickened intimta.

Cholesterol crvstals were fouind onilv in thecoronary lesions of the omnivorous animals(chimpanzees, panida). The lesion-is of the vege-tarian vertebrates, either miaminlals (niioufflon,tapir) or birds (crane, free (lucks). wTere quiitefree froni this sterol.

Diffuse intimnal sclerosis is a much. miiorecomnion finding thani atheroseler osis in thecoronarv arteries we investigated: it was ob-'served in more than 25 per cent of the eaptivet

Circulation, Volumte XXVI, November 1962

846

NATI'RIAL 11FTOIIY ( ti' AIIElIiOSQLEIlO8ITS

A

Figure 4NItherloserotbe plaque in the ri ht Ioronaryr(Jrer/fof a tapir. Lipid infiltration owas pres(-nt in theother coronary trunks of this animal. 1eigefrt:X 10.

hinalll]Wds 1(i nearix 1 p rent ofdthfebir'1ds. il the ecaptiVe ver.teltrates of our11 innW-terial, the freqnevey ()of liffinse intiual scle-lSis se(1ems to be hlig.h.e.r amlong01' omlnil;vorouIsandlherbi-vorous aininals than a.mong' earnruv-orous annua.ls.

Amnong- primates, 52) per ecuit of whici slhowvt1mo0re Or less 1i1mportnIt; fibrolls thickeIing1 Ofthe iuitina in their eoroniary arteries, the lessvu-lnierable to itimntal sclerosis wvere ltlelen-iidae, anid the mo-s Auileraievwere thwe pol-gidae (table 5).

Although1) nio sign. of coroarvy atherosclero-sis couldl l)e diseoTered ini annualmIs shot in-freedoi, the coronarr-v arteries of deer, roe-buck, u ild boar, mnellys, anmd chimpanzeesoften shiowved diffuLse intimlal selerosis, w-hielappqwarently j-intensifiedI wvith age. This is, howv-ever, very difficlLt to estimiiate because thevari.ations ini the vulnierabilitv to intimalsclerosis are niot only specific. but also idi-vidnal.

TI every case, ehimpaizees alI grorillas(andl, to a less degree, orag-utans) are witlhmi-an the onily species of thie atfnim-l kiigrdomiin wic-leih dliftnse intfimal selerosis is a genleral-ize(d featuire antIi begins as early as the firstweeks of life.

Of iiteiest iv as the diseoverv of ani organ-izingo thiroimibus in the right eoronary arteryof a 7-month-old boar that was hunted and(-(irculation, Volatnoc XXVI, Norcmber 1.962

IT..

Figure nCoronary thrombosis lin a 7-month-old :wild, boar.[The thr-ombuzs deIclopedi in the righlt coronary!ar-terq in spite of the absentce of atherosclerosisin the coronitary arilter-ial tree a1wl( in1 tlhe aorta.WcVigert: A, X 2.t5; B, X .10.

slolt. (fig. 5) :in thlis animial, throimibosis de1,-Velopled on pire initimyial sclerosis, in spite of'the v\lerified absence of any lipid deposit eith-elrin the aorta or in t1e coroniary arterial tree.

Biochemical ResultsTotal Cholesterol anid Phospholipids

Thle level of totail cholesterol is by nio neanisecharacteristie of the species. The range iscomiiprised betw-eeni 7.5 aIndi 250 nig. per ceniitfor., whole mnateriail. Triel.e are nio strikingyn,genierie differences in. t lie clholesterol /plosplio-lipid iratio, amiong captilVe orl amllloln freespecimnes (old )looCled, 0.56 to 0.9; carntiv-orIols. 0.50 to 0.60( vegetariani ande onnfiv-or-(Ins. 0.70 to 1.12). indilviduil variationis max-lie great (table 6).

847

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VASTESAEGER, DELCOIRT

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VASTESAEGER, DELCOURT

CAAN,VOautL

PRI tATE4Figure 6

Evolution of the lipidogram in different groups ofmammals. Arrow indicates: Left. Liptidographicpattern in primates; (top) cercopithecidae, (cen-ter) galago (ga'lagidae%, (bottonm) potto (lorisi-dae.). Center. Lipidographic pattern in baboons;in freedom (straight line), in captivity (dottedline). Right. Lipidographic pattern in (top) ungu-

lates, (center) carnivora, (bottom) pholidota.

Whole Lipoproteins

The lipidogram pattern is more characteris-tic of the species. As a rule, the lipidogramseems to be related to evolution: the more

evoluted the species, the more the alpha-lipo-protein fraction becomes prominent (fig. 6).

Isolation and Characterization of Beta-LipoproteinsBurstein"5 described a method suitable for

quantitative separation of beta-lipoproteins.Dextran sulfate of high molecular weight anda molar solution of caleium chloride are addedto the serum. Beta-lipoproteins are by thisway selectively precipitated. But even in hu-man serum, there is a rather strict dependencebetween the precipitation of beta-lipoproteinsand the molecular weight of dextran sulfate.18The procedure of Burstein remains question-able, however, when one tries to study lipo-proteins in animals other than man.

I

Figure 7Lipidograms of a wild boar: I. in freedom; II., incaptivity.

Lipidograms confirm that the beta-lipopro-teins are correctly separated by this method,in cold-blooded vertebrates as well as in mam-mals. However, Sakagami and Zilversmit,9have shown that dextran sulfate is a suitablereagent to separate quantitatively the beta-lipoproteins of the dog serum.

In our experience, the precipitation is notlinked with any antigenic property of lipo-proteins. Using antihuman beta-lipoproteinserum, we found fractions having a commonantigenicity with human fraction only inmonkeys and in some antelopes (Cephalophusmonticola [Thunb] ).

Generally speaking, as in human serum, thepercentage of cholesterol bound to beta-lipo-proteins in wild animals was not very differ-ent from the percentage of this fraction asdemonstrated by lipidogram. But absolutelevels were quite dispersed. Extensive studiesshould be done on many individuals in eachspecies before one may even attempt to sys-tematize the biochemical results and the spe-cific trend toward atherosclerosis (table 7).

Lipoproteins and CaptivityIt can be seen, either by lipidograms or by

chemical determinations, that captivity has adeleterious effect on the beta-lipoprotein lev-els. In lipidograms, the beta peak becomesvery prominent; the total cholesterol is muchhigher, as is the level of cholesterol bound tobeta-lipoproteins (fig. 7, table 7). The changes


85Q''

-WC i ccct. I.L 'a. t


Table 7Cholesterol and Phospholipid Levels and Ratios

Species

Wild boar (Sus se

Pangolins(Manis tricuspis)

Mangabeys(Cereocebus ateri

Baboons*(Papio doguera)

Status

arofa)

12

I1 Freedom2 Captivity, Europe3 Captivity, Europe

FreedomCaptivity, Congo

imus) 1 Captivity, Congo2 Capitivity, Europe3 Captivity, Europe

1 Captivity, Congo2 Captivity, Congo3 Freedom4 Freedom5 Freedom6 Freedom

CholesterolTotal a 18 % ,

71 51 20 28114 104 10 9193 139 54 27

145 110 35220 97 133

127204211

17514214415717070

527892

2620

5811355

75125119

149122

995716

2460

606157

8587

633327

PhospholipidsTotal a 18

176 84 92156 119 37144 100 44

256 218 38 1.08243 160 83 .62

160256202

214158

212272160

1187373

10082

11814673

42189169

.561.461.08

11476

9412687

.77

.62

.952.25.4

*Tn spite of slight specific differences, our figuresfound in Kenya baboons living in freedom, as wellcaptive specimens of Papio ursinus.

in the phospholipids, either total or bound tobeta-lipoproteins, are less pronounced.

DiscussionUntil now, most investigations concerning

spontaneous degenerative processes have beencarried out either on domestic animals or on

captive wild animals. Of course the domesticones have been deeply modified in their mor-

phology as well as in their physiology by theartificial selection that man has brought tobear on them, sometimes during thousands ofyears.

One must then ask if it is not human in-fluence that, either by domestication or bycaptivity, has favored the birth and the de-velopment of the arterial degenerative proc-esses observed in the examined animals. Inorder to answer this question, one should beable to compare under rigorous conditions thearterial state of animals of the same species,captive and in their wild state. First of all,in view of the density of human populationand its dispersion over all the lands of theglobe, and also of the multiplicity and power

of the commanding means of man over nature,it is difficult to conceive that there could still

are in agreement with those of McGill and co-workers'0as with those of Van Zyl and Kerrick' obtained in

exist animals that entirely escape human in-fluence. If such conditions still exist, theyare realized in the sea: the discovery of in-timal sclerosis and of atherosclerosis in tunnyfishes caught in their natural habitat bringsproof that the two processes are not a con-

sequence of captivity in the case of cold-blooded vertebrates.As far as warm-blooded animals are con-

cerned, there are still wild specimens livingin places almost inaccessible to man and hav-ing only brief and incidental contacts withhim, that have kept to strictly natural food-stuffs and to a natural way of life. In orderto establish the influence of captivity on thegenesis and evolution of arteriosclerosis, therestill remains the difficulty of procuring indi-viduals of the same race that are comparableas far as age, sex, and stage of developmentare concerned. All these conditions are farfrom being fulfilled in our material: this iswhy we cannot draw any firm conclusionconcerning the influence of captivity on theincidence and evolution of intimal sclerosisand atherosclerosis in birds and mammals.

Nevertheless, the lesions we observed in

Circulation, Volume XXVI. November 1962

,8 Phospholipids13 Cholesterol

4.63.7.81

851


deer, in colobi, and in chimpanzees living infreedom bring evidence that, at least in rumi-nants and in primates, neither intimal scle-rosis nor atherosclerosis is a mere consequenceof captivity. From the comparison of sectionsmade in the same coronary trunk of youngcaptive chimpanzees and of free chimpanzeesnearly of the same age, we got the impressionthat the importance of individual variationsin the stage of intimal sclerosis in this speciesmay occasionally surpass the influence of cap-tivity on this process.Our biochemical results are also rather

disappointing for the study of the naturalhistory of atherosclerosis. Before the resultsare discussed, the following points should beemphasized. 1. Even in freedom, an animalpresumed normal is not necessarily normal.Specimens shot or trapped in their habitatare usually disabled or ill, the healthy andfit ones escaping more easily. In any case, wehad the opportunity to see how severely trop-ical animals are infested with parasites (table6); unfortunately the influence of parasiteson the lipoproteins pattern is still unknown.2. Captive animals are not normal. Two kindsof captivity are to be considered: in the firstone, the animals remain in their natural cli-mate, not far away from their original habi-tat; in the second one, the animals are sentto zoos in far distant countries. In the firstcase their natural feeding grounds are littledisturbed, but in the second case diet andenvironmental conditions are quite different.As compared with the values recorded in

freedom, both types of captivity significantlyalter lipoproteins as well as cholesterol levels.This was observed not only in European sui-dae and carnivora, but also in African un-gulates and primates. This deleterious effectof captivity is impressively confirmed by apreviously published study of 60 young chim-panzees living for many months at the medicalLaboratory of Stanleyville.22 Table 8 sum-marizes the results of this study. The totalblood cholesterol ranged between 120 and 470mg. per cent, the mean value being 250 mg.per cent. In all specimens the cholesterol

fraction bound to the beta-lipoproteins wasquite large. No relationship between choles-terol level and age, sex, or length of captivitycould be established.

In any case, our biochemical results bringaccumulated evidence that captivity may sig-nificantly alter some aspects of the lipidicmetabolism in animals. This should be keptin mind when one seeks suitable material forexperimental atherosclerosis.As far as comparative arterial pathology

is concerned, our observations confirm thoseof Fox dealing with the widely separatedvarieties of the affected vertebrates. They alsoconfirm the statement, at first made by Fox,that the lesions are mainly distributed in theaorta, the great majority of the involvedspecimens showing this distribution alone.Our results disagree, however, with those ofFox concerning frequency and specific inci-dence of atherosclerosis: in Fox 's material"lipidic deposits were exceedingly rare," inours, nearly 30 per cent of the examined aor-tas were affected. On the other hand, in Fox'smaterial, primates were less vulnerable thancarnivora and ungulates to degenerative arte-rial disease: on the contrary, it was amongprimates that we found the highest percentageof aortic lesions. Such a discrepancy can ofcourse be explained by the; difference of con-ditions of captivity in the zoos of Antwerpand Philadelphia.

Different local conditions of diet are prob-ably not the only factors responsible for thediscrepaney between Fox's figures and ours:in the same Zoo of Philadelphia, between 1931and 1956, Ratcliffe and Cronin7 observed aninereasing frequency of arteriosclerosis incaptive wild animals, independent of age, anddiet.The results of our investigations are very

difficult to compare with the figures publishedby Rateliffe and Cronin. As a matter of fact,the criteria of these authors are quite differ-ent from ours: their practice has been "toignore isolated plaques of the aorta and largerbranches, unless they were accompanied byvascular disease of the heart, kidney or

Circulation, Volume XXVI, November 196.t

852

NATURAL IIISTORtY OF ATIJEROSULEROSIS 853

sptleen.' Furithermiore. tlhey imla-de 110 elih-e(late effort to separate atherosclerosis fromotler formus of arterioselerosis.The numiber of our specimenes is, of course,

too small to allowv even a ronghi estimiiate ofthe frequenev of both intiinal sclerosis andatheroselerosis in eaeli speeies of vertebrates.But7 as far as the incidence of spontaneousatheroselerosis is concerned, w-e have beenstruck by its greater frequlency amiong cer-tamn species stueh as tree duekls (three of fiveaninnials had eoronary atherosclerosis) andamnono,g cenera like cercopithecidae anid al)es.The frequeney of atheroselerosis is hig,her inthe prima-tes thani in the ungrulates, and hiigherin the ungulates than in the carnivora.On tile othe-r lhand, in its hIman incidence

diffuise intinial selerosis appears as a general-ize(1 process, beginnling as soonl as the firstAveeks of life in white as wcll as in Bantupeople?3 Such universal preeoeity among h-i111811 beiniis gave manv autlhors the imnpressionithat diffuise intinual selerosis could be a phys-iologic proeess, growingf progressively withadvancingf agre. AAWhereas intimal seletosis isfoullnd. in all voungr human aduilts, this is farfi-oun the ease in other species.

-Although the facts brought about by our11iinvYesticgations are very fragmentary, tley arequtiite sufficienit to establishl that not onilv theineidenee of diffuse intimal sclerosis but alsothe age at which it appears vary eonisiderablvfrom one speeies to another. Obviously, thefact that we cannot know the real agfe of cap-tive animals makes any kind of precisiondifficult, especially if the variable life spanof the different species is taken inito account.Nevertheless, the faet that the coronary ar-

teries of a captive wild pig, which died ofold age (fig. 8, top), show hardly ml-ore ad-vaniced lesions than those of a 5-year-oldgorilla (fig. 8, bottom) proves undeniably theexistence of specific differences in the precoc-ity of the fibrous thickeniing of the coroaiavyintima. We could multiply suci examiples Candshow that of all mrlammals the anthropoid apesand mrlanl are the only ones to present systemn-Circulation, Volutme XXVI, November 1962

Figure 8'1'oj. Anterior descending branch of an o1(1 cO]pti 1

wart-hog. The internal elastic nembran ivstretched and ruptured, bat thte jatiima is moloeiE-ately thickened. lVeigert X 40. Bottoml. Anteriordesceniding branch of a ylounignInmature fecna7gorilla. Tfhe internal elastic membrane is as thickas that in the wart-hog.

atically importanit fibrous changtes of the cor-

onary iintima before puberty.The larger number of primnates we could

investigfate gave us the impression that in thisor(ler at least the frequency and the precocityof initiial fibrosis increase with the degree ofevolution (table 5).

In ouir experienee, spontaneous atherosecie-rosis always develops in arteries alreadly im-paired by iintimal selerosis. In otlher vwords,iintimal sclerosis appears as a basic prelim-ilmary lCondition for spontanieous atherogenesis.To what extent alterations of lipidi netab-

olism facilitate spontaneous atherosilerosis inithie fibirous initiina is still unikniowin, btut it isfirllliy established tihat atherosclerosis may

853


spontaneously develop in animals having quitedifferent diets, and especially in fishes feed-ing on a diet rich in unsaturated fats; incaptive birds whose dietary fats are mostlyunsaturated (crane, tree duck, pelican) ; inwild mammals whose diet is free of animalfats, in freedom as well as in captivity (deer,camel, tapir).

These facts indicate that there is no abso-lute and uniform relationship-i.e., applicableto every vertebrate species and to every indi-vidual in a given species between the originor the chemical nature of dietary fats andthe development of atherosclerosis. Inbredspecific and individual differences in vulner-ability to intimal sclerosis, reinforced byspecific and individual differences in enzy-matic activity of lipid metabolism, yield abetter explanation of the great variability inthe incidence of atheroselerosis in differentspecies and in different individuals of thesame species. Captivity or way of life (diet,physical activity, and stress) are further var-iable elements in the problem of atherogenesisin wild animals.

SummaryIn order to complete the fragmentary

knowledge of the natural history of athero-sclerosis, the authors examined the coronaryarteries of 403 captive and 146 wild verte-brates living in freedom, as well as the aortasof 134 of them. In 88 of the captive animalsand in 51 of the free individuals, lipidogram,total cholesterol, beta-cholesterol and choles-terol/phospholipid ratio were also determined.

In this material, spontaileous atherosclerosisalways developed in arteries already impairedby intimal selerosis, and intimal sclerosisseems to be a basic condition for spontaneousatherogenesis.

Lipid deposits were found in the intima of29 per cent of the examined aortas, but cor-onary atherosclerosis was present in only 2per cent of the captive specimens. The dis-covery of coronary atherosclerosis in freetunny fishes, caught in the Bay of Biscay,brings proof that atheroselerosis is neither

the privilege of warm-blooded vertebrates nora consequence of captivity.

Unfortunately, the material collected doesnlot permit even a rough estimate of the in-fluence of captivity on the development ofatherosclerosis. It was observed in any casethat captivity significantly alters some aspectsof the lipidic metabolismi in animals, andespecially the beta-lipoprotein levels as wellas the beta-cholesterol fraction.

Spontaneous atherosclerosis may develop infishes living in their natural habitat, feedingon a diet rich in unsaturated fats; in captivebirds whose dietary fats are mostly unsat-urated (crane, tree duck, pelican) ; and inwild mammals whose diet is free of animalfats, in freedom as well as in captivity (deer,eamel, tapir).

References1. LOBSTEIN, J. G. C. F. M.: Traite d 'anatomie

pathologique. Paris, Levrault, 1833.2. M6NCKEBERG, J. G.: t;ber die reine Mediaver-

kalkung der Extreiitiitenarterien urnd ihr Ver-halten zur Arteriosklerose. Virchows Arch. f.path. Anat. 171: 141, 1903.

3. World Health Organization: Rapport d 'un grouped 'etude: Classification des lesions d 'athero-selerose. Palais des Nations, Geneve, 1958.

4. MARCHAND, F.: uber Arterioskierose (Athero-Sklerose). Verhandl. (T. 21. Kong. F. inn. Med.21: 23, 1904.

5. Fox, H.: Disease in Captive Wild Mammalsand Birds. Philadelphia, J. B. Lippincott Com-pany, 1923.

6. Fox, H.: In Cowdry, E. V.: Arteriosclerosis,a Survey of the Problem. New York, TheMacmillan Company, 1933, p. 153.

7. RATCLIFFE, H. L., AND CRONIN, 1. T.: Changingfrequency of arteriosclerosis in mammals andbirds at the Philadelphia Zoological Garden.Circulation 18: 41, 1958.

8. RATCLIFFE, H. L., YERASIMIDEs, T. G., ANDELLIOTT, G. A.: Changes in the character andlocation of arterial lesions in mammals andbirds in the Philadelphia Zoological Garden.Circulation 21: 730, 1960.

9. DAUBER, D. V.: Spontaneous arteriosclerosis inchickens. Arch. Path. 38: 46, 1944.

10. BRAGDON, J. H.: Spontaneous atherosclerosis inthe rabbit. Circulation 5: 641, 1952.

11. LINDSAY, S., CHAIKoFF, I. L., AND GILMORE,J. L.: Arteriosclerosis in the dog: Spontaneouslesions in the aorta and the coronary arteries.Arch. Path. 53: 281, 1952.

Circulation, Volume XXVI, November 196.2

854


12. LINDSAY, S., CHAIKOFP, I. L., AND GILMORE, J. L.:Arteriosclerosis in the cat: Naturally occurringlesions in the aorta and the coronary arteries.Arch. Path. 40: 29, 1955.

13. LOPLAND, H. B., AND CLARKSON, T. B.: A bio-chemical study of spontaneous atherosclerosisin pigeons. Circulation 7: 234, 1959.

14. HAHN, P. F.: Abolishment of alimentary lipemiafollowing injection of heparin. Science 98:19, 1943.

15. DELCOURT, R.: Les extrapolations cliniques desstatistiques biologiques. I. Resultats d'une en-

quete sur la cholesterolemie. Acta dlin. belg.15: 26, 1960.

16. ROBERTSON, 0. H., WEXLER, B. C., AND MILLER,B. F.: Degenerative changes in the cardio-vascular system of the spawning Pacific salmon(Oncorhynchus tshawytscha). Circulation Re-search 9: 826, 1961.

17. VASTESAEGER, M., GILLOT, P. H., AND MORTEL-MANS, J.: Thrombose coronarienne ayantentraine la mort chez un panda (Ailurusfulgens). Bull. Soc. roy. Zool. Anvers 14: 47,1959.

18. BURSTEIN, M.: Sur une nouvelle m6thode dedosage des lipoprotienes du serum. Path. etbiol. 6: 541, 1958.

19. SAKAGAMI, T., AND ZILVERSMIT, D. B.: Separa-tion of dog serum lipoproteins by ultracentri-fugation, dextran sulfate precipitation andpaper electrophoresis. Lipid Research 2: 271,1961.

20. MCGILL, H. C., STRONG, J. P., HOLMAN, R. L.,AND WERTHESSEN, N. T.: Arterial lesionsin Kenya baboon. Circulation Research 8: 670,1960.

21. VAN ZYL, A., AND KERRICK, J. E.: Serum lipidsand age in the baboon (Papio ursinus).South African J. M. Sc. 20: 97, 1955.

22. VASTESAEGER, M., AND DELCOURT, R.: Spon-taneous atherosclerosis and diet in captiveanimals. Nutritio et dieta 3: 174, 1961.

23. ZANCHI, D. AI., GILLOT, P. H., AND RASSON, G.:Morphologie des arteres coronaires dans lapremiere enfance chez le blanc et le bantou.2eme Colloque International sur la morbiditecoronarienne dans le monde: Rome, 1960.Cardiologia Pratica 13: 147, 1962.

Men of science who mean to embrace the principles of the experimental method as a

whole, must fulfill two classes of conditions and must possess two qualities of mindwhich are indispensable if they are to reach their goal and succeed in the discovery oftruth. First, they must have ideas which they submit to the control of facts; but atthie same time they must make sure that the facts which serve as starting point or as

control for the idea are correct and well established; they must be at once observersand experimenters.-CLJAUDE BERNARD. An Introduction to the Study of ExperimentalMedicine. New York, The Macmillan Company, 1927, p. 21.

Circukation, Volume XXVI, November 1962

855

the natural history of atherosclerosis

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